Anticancer compounds, pharmaceutical compositions thereof, and a method of treating cancer

ABSTRACT

Cytotoxic compounds containing a phenyl core, amide link(s), an imidazolinone or a propenamide moiety. Also described are pharmaceutical compositions incorporating the cytotoxic compounds and methods for treating cancer. These compounds are cytotoxic against breast, prostate, and leukemia cancer cell lines via dual inhibition of Src kinases and tubulin.

STATEMENT OF FUNDING ACKNOWLEDGEMENT

This project was funded by the Deanship of Scientific Research (DSR),King Abdulaziz University, Jeddah, the Kingdom of Saudi Arabia, undergrant number RG-4-166-40.

BACKGROUND OF THE INVENTION Technical Field

The present disclosure relates to compounds with anti-proliferativeactivity and a method of treating cancer.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

The abnormal expression of protein tyrosine kinases (PTK) leads to cellproliferation disorder and is associated with tumor invasion,metastasis, and angiogenesis. As a result, a variety of PTKs have beenused as targets for screening anti-tumor drugs [Drake, J. M.; Lee, J.K.; Witte, O. N., Clinical targeting of mutated and wild-type proteintyrosine kinases in cancer. Molecular and cellular biology 2014, 34(10), 1722-32].

Currently, all PTK inhibitors that are clinically available are thosethat occupy ATP pockets. Since ATP is a common cofactor that isinherently concentrated in cells (mM), large quantities of the inhibitormust be delivered to the ATP pockets to achieve adequate selectivity andaffinity. A practical, though under-utilized approach, is to developkinase inhibitors that bind to the substrate site. This approach shouldbe appealing because each kinase is specific for a unique peptidesequence.

The Src family of kinase enzymes (SFK) are non-receptor tyrosine kinasesessential in signaling machinery [Guo, W.; Giancotti, F. G., Integrinsignalling during tumour progression. Nature reviews Molecular cellbiology 2004, 5 (10), 816]. Members of SFKs include Src, Yes, Fyn, Fgr,Lck, Hck, Blk, Yrk, and Lyn. SFKs are important for fundamental cellularprocesses such as cell growth, functions, survival, proliferation,differentiation, and migration [Lieu, C.; Kopetz, S., The SRC family ofprotein tyrosine kinases: a new and promising target for colorectalcancer therapy. Clinical colorectal cancer 2010, 9 (2), 89-94]. Aberrantactivities of SFK have been linked to a variety of cancers includingthose of the prostate, breast, colon, lungs, pancreas, brain,melanocytes, and bone marrow [Frame, M. C.; Roskoski, R., Src FamilyTyrosine Kinases. In Reference Module in Life Sciences, Elsevier: 2017].Inhibitors of Src kinase such as dasatinib, bosutinib, saracatinib,KX-01, and KX-02 have been recently developed for cancer treatment[Elsberger, B.; Stewart, B.; Tatarov, O.; Edwards, J., Is Src a viabletarget for treating solid tumours? Current cancer drug targets 2010, 10(7), 683-694; Rothschild, S. I.; Gautschi, O.; Haura, E. B.; Johnson, F.M., Src inhibitors in lung cancer: current status and future directions.Clinical lung cancer 2010, 11 (4), 238-42; and Smolinski, M. P.; Bu, Y.;Clements, J.; Gelman, I. H.; Hegab, T.; Cutler, D. L.; Fang, J. W. S.;Fetterly, G.; Kwan, R.; Barnett, A.; Lau, J. Y. N.; Hangauer, D. G.,Discovery of Novel Dual Mechanism of Action Src Signaling and TubulinPolymerization Inhibitors (KX2-391 and KX2-361). Journal of medicinalchemistry 2018, 61 (11), 4704-4719, each incorporated herein byreference in their entirety]. Despite these recent efforts, there isstill a need to develop more effective non-ATP competitive inhibitors asanti-proliferative agents.

In view of the forgoing, one objective of the present disclosure is toprovide therapeutic compounds with anticancer activities, apharmaceutical composition comprising thereof, and a method for cancertreatment.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to acompound selected from the group consisting of a compound of formula(I),

a salt thereof, a solvate thereof, a tautomer thereof, and astereoisomer thereof;a compound of formula (II),

a salt thereof, a solvate thereof, a tautomer thereof, and astereoisomer thereof;a compound of formula (III),

a salt thereof, a solvate thereof, a tautomer thereof, and astereoisomer thereof; and mixtures thereof, wherein (i) R₁, R₁′, and R₁″are independently selected from the group consisting of an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, and an optionally substituted aryl, (ii) R₂, R₂′,and R₂″ are independently an optionally substituted aryl, or anoptionally substituted heteroaryl, (iii) R₃ and R₃′ are independently anoptionally substituted aryl, or an optionally substituted heteroaryl,(iv) R₄ and R₄′ are independently selected from the group consisting ofa hydrogen, an optionally substituted alkyl, an optionally substitutedcycloalkyl, an optionally substituted arylalkyl, and an optionallysubstituted aryl, (v) R₅, R₅′, R₆, and R₆′ are independently selectedfrom the group consisting of a hydrogen, an optionally substitutedalkyl, an optionally substituted cycloalkyl, and an optionallysubstituted arylalkyl, (vi) R₇, R₇′, R₇″, R₈, R₈′, R₈″, R₉, R₉′, R₉″,R₁₀, R₁₀′, R₁₀″, and R₁₁ are independently selected from the groupconsisting of a hydrogen, an optionally substituted alkyl, an optionallysubstituted cycloalkyl, an optionally substituted arylalkyl, a hydroxy,and a halogen, and (vii) m and n are independently an integer in a rangeof 1-3.

In one embodiment, the compound is represented by formula (I), or a saltthereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₁ is methyl, or phenyl.

In one embodiment, the compound is represented by formula (I), or a saltthereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₂ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, and2-furanyl.

In one embodiment, the compound is represented by formula (I), or a saltthereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₃ is phenyl.

In one embodiment, the compound is represented by formula (I), or a saltthereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₄ is hydrogen.

In one embodiment, the compound is represented by formula (I), or a saltthereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₅ and R₆ are hydrogen.

In one embodiment, the compound is represented by formula (II), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₁′ is methyl, or phenyl.

In one embodiment, the compound is represented by formula (II), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₂′ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, andp-fluoro-o-methylphenyl.

In one embodiment, the compound is represented by formula (II), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₃′ is selected from the group consisting of phenyl,p-methoxyphenyl, 2-furanyl, p-fluorophenyl, and 4-1,1′-biphenyl.

In one embodiment, the compound is represented by formula (II), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₄′ is hydrogen or methyl.

In one embodiment, the compound is represented by formula (II), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₅′ is hydrogen, and R₆′ is hydrogen, or methyl.

In one embodiment, the compound is represented by formula (III), or asalt thereof, a solvate thereof, a tautomer thereof, or a stereoisomerthereof, and R₁″ is methyl, R₂″ is phenyl, and R₁₁ is methyl.

In one embodiment, R₇, R₇′, R₇″, R₈, R₈′, R₈″, R₉, R₉′, R₉″, R₁₀, R₁₀′,and R₁₀″ are hydrogen.

In one embodiment, the compound is selected from the group consisting of

According to a second aspect, the present disclosure relates to apharmaceutical composition, comprising the compound of the first aspectand a pharmaceutically acceptable carrier and/or excipient.

In one embodiment, the pharmaceutically acceptable carrier and/orexcipient is at least one selected from the group consisting of abuffer, an inorganic salt, a fatty acid, a vegetable oil, a syntheticfatty ester, a surfactant, and a polymer.

In one embodiment, the compound is selected from the group consisting of

According to a third aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringthe pharmaceutical composition of the second aspect to a subject in needof therapy.

In one embodiment, 0.1-500 mg/kg of the compound is administered perbody weight of the subject.

In one embodiment, the proliferative disorder is cancer, and the canceris at least one selected from the group consisting of breast cancer,prostate cancer, and leukemia.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a schematic summary of the design of compounds of the presentdisclosure.

FIG. 2 shows a list of KIM-C compounds.

FIG. 3 shows a list of KIM-V compounds.

FIG. 4 is a scheme illustrating the synthesis of amine startingmaterials 4a-j.

FIG. 5 is a scheme illustrating the synthesis of KIM-C and KIM-Vcompounds.

FIG. 6A shows the cytotoxicity of compound KIM-161C against MCF7 breastcancer cells.

FIG. 6B shows the cytotoxicity of compound KIM-241C against MCF7 breastcancer cells.

FIG. 6C shows the cytotoxicity of compound KIM-221V against MCF7 breastcancer cells.

FIG. 7A shows the cytotoxicity of compound KIM-131C against PC3 prostatecancer cells.

FIG. 7B shows the cytotoxicity of compound KIM-161C against PC3 prostatecancer cells.

FIG. 7C shows the cytotoxicity of compound KIM-241C against PC3 prostatecancer cells.

FIG. 7D shows the cytotoxicity of compound KIM-221V against PC3 prostatecancer cells.

FIG. 8 is an overlay of dose-response curves of HL-60 leukemia cellsupon treatment with increasing concentrations of compounds KIM-161C andKIM-241C.

FIG. 9A is a bar graph summarizing the effect of compound KIM-161C ondifferent cell cycle stages of HL-60 leukemia cells.

FIG. 9B shows different cell cycle stages of HL-60 leukemia cells upontreatment of blank control (i.e. cells grown in the absence of anycompound).

FIG. 9C shows different cell cycle stages of HL-60 leukemia cells upontreatment of KIM-161C at a concentration of 362.5 nM.

FIG. 9D shows different cell cycle stages of HL-60 leukemia cells upontreatment of KIM-161C at a concentration of 725 nM.

FIG. 10A is a bar graph summarizing the effect of KIM-161C on apoptosisin HL-60 leukemia cells.

FIG. 10B is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of blank control (i.e. cells grown in the absenceof any compound) for 24 hours.

FIG. 10C is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of KIM-161C at a concentration of 362.5 nM for 24hours.

FIG. 10D is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of KIM-161C at a concentration of 725 nM for 24hours.

FIG. 10E is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of blank control (i.e. cells grown in the absenceof any compound) for 48 hours.

FIG. 10F is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of KIM-161C at a concentration of 362.5 nM for 48hours.

FIG. 10G is a histogram showing Caspase-3 activity of HL-60 leukemiacells upon treatment of KIM-161C at a concentration of 725 nM for 48hours.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

As used herein, the words “a” and “an” and the like carry the meaning of“one or more”. Within the description of this disclosure, where anumerical limit or range is stated, the endpoints are included unlessstated otherwise. Also, all values and subranges within a numericallimit or range are specifically included as if explicitly written out.

As used herein, the terms “compound”, “starting material”, and “product”are used interchangeably, and are intended to refer to a chemicalentity, whether in the solid, liquid or gaseous phase, and whether in acrude mixture or purified and isolated.

As used herein, the term “solvate” refers to a physical association of acompound of this disclosure with one or more solvent molecules, whetherorganic or inorganic. This physical association includes hydrogenbonding. In certain instances, the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. The solvent molecules in thesolvate may be present in a regular arrangement and/or a non-orderedarrangement. The solvate may comprise either a stoichiometric ornonstoichiometric amount of the solvent molecules. Solvate encompassesboth solution phase and isolable solvates. Exemplary solvents include,but are not limited to, water, methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, tert-butanol, ethyl acetate andother lower alkanols, glycerine, acetone, dichloromethane (DCM),dimethyl sulfoxide (DMSO), dimethyl acetate (DMA), dimethylformamide(DMF), isopropyl ether, acetonitrile, toluene, N-methylpyrrolidone(NMP), tetrahydrofuran (THF), tetrahydropyran, other cyclic mono-, di-and tri-ethers, polyalkylene glycols (e.g. polyethylene glycol,polypropylene glycol, propylene glycol), and mixtures thereof insuitable proportions. Exemplary solvates include, but are not limitedto, hydrates, ethanolates, methanolates, isopropanolates and mixturesthereof. Methods of solvation are generally known to those of ordinaryskill in the art.

As used herein, the term “tautomer” refers to constitutional isomers oforganic compounds that readily convert by tautomerization ortautomerism. The interconversion commonly results in the formalmigration of a hydrogen atom or proton, accompanied by a switch of asingle bond and adjacent double bond. Tautomerism is a special case ofstructural isomerism, and because of the rapid interconversion,tautomers are generally considered to be the same chemical compound. Insolutions in which tautomerization is possible, a chemical equilibriumof the tautomers will be reached. The exact ratio of the tautomersdepends on several factors including, but not limited to, temperature,solvent and pH. Exemplary common tautomeric pairs include, but are notlimited to, ketone and enol, enamine and imine, ketene and ynol, nitrosoand oxime, amide and imidic acid, lactam and lactim (an amide and imidictautomerism in heterocyclic rings), and open-chain and cyclic forms ofan acetal or hemiacetal (e.g., in reducing sugars).

As used herein, the term “stereoisomer” refers to isomeric moleculesthat have the same molecular formula and sequence of bonded atoms (i.e.constitution), but differ in the three-dimensional orientations of theiratoms in space. This contrasts with structural isomers, which share thesame molecular formula, but the bond connection of their order differs.By definition, molecules that are stereoisomers of each other representthe same structural isomer. Enantiomers are two stereoisomers that arerelated to each other by reflection, they are non-superimposable mirrorimages. Every stereogenic center in one has the opposite configurationin the other. Two compounds that are enantiomers of each other have thesame physical properties, except for the direction in which they rotatepolarized light and how they interact with different optical isomers ofother compounds. Diastereomers are stereoisomers not related through areflection operation, they are not mirror images of each other. Theseinclude meso compounds, cis- and trans- (E- and Z-) isomers, andnon-enantiomeric optical isomers. Diastereomers seldom have the samephysical properties. In terms of the present disclosure, stereoisomersmay refer to enantiomers, diastereomers, or both.

Conformers, rotamers, or conformational isomerism refers to a form ofisomerism that describes the phenomenon of molecules with the samestructural formula but with different shapes due to rotations around oneor more bonds. Different conformations can have different energies, canusually interconvert, and are very rarely isolatable. There are somemolecules that can be isolated in several conformations. Atropisomersare stereoisomers resulting from hindered rotation about single bondswhere the steric strain barrier to rotation is high enough to allow forthe isolation of the conformers. In terms of the present disclosure,stereoisomers may refer to conformers, atropisomers, or both.

In terms of the present disclosure, stereoisomers of the double bonds,ring systems, stereogenic centers, and the like can all be present inthe compounds, and all such stable isomers are contemplated in thepresent disclosure. Cis- and trans- (or E- and Z-) stereoisomers of thecompounds of the present disclosure wherein rotation around the doublebond is restricted, keeping the substituents fixed relative to eachother, are described and may be isolated as a mixture of isomers or asseparated isomeric forms. S- and R- (or L- and D-) stereoisomers of thecompounds of the present disclosure are described and may be isolated asa mixture of isomers or as separated isomeric forms. All processes ormethods used to prepare compounds of the present disclosure andintermediates made therein are considered to be part of the presentdisclosure. When stereoisomeric products are prepared, they may beseparated by conventional methods, for example, by chromatography,fractional crystallization, or use of a chiral agent.

As used herein, the term “substituted” refers to at least one hydrogenatom that is replaced with a non-hydrogen group, provided that normalvalencies are maintained and that the substitution results in a stablecompound. When a substituent is noted as “optionally substituted”, thesubstituents are selected from halo, hydroxyl, alkoxy, oxo, alkanoyl,aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino,disubstituted amines (e.g. in which the two amino substituents areselected from the exemplary group including, but not limited to, alkyl,aryl or arylalkyl), alkanoylamino, aroylamino, aralkanoylamino,substituted alkanoylamino, substituted arylamino, substitutedaralkanoylamino, thiol, alkylthio, arylthio, arylalkylthio, alkylthiono,arylthiono, aryalkylthiono, alkylsulfonyl, arylsulfonyl,arylalkylsulfonyl, sulfonamide (e.g. —SO₂NH₂), substituted sulfonamide,nitro, cyano, carboxy, unsubstituted amide (i.e. —CONH₂), substitutedamide (e.g. —CONHalkyl, —CONHaryl, —CONHarylalkyl or cases where thereare two substituents on one nitrogen from alkyl, aryl, or alkylalkyl),alkoxycarbonyl, aryl, substituted aryl, guanidine, heterocyclyl (e.g.indolyl, imidazoyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl,pyrimidiyl, pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl,homopiperazinyl and the like), substituted heterocyclyl and mixturesthereof. The substituents may themselves be optionally substituted, andmay be either unprotected, or protected as necessary, as known to thoseof ordinary skill in the art, for example, as taught in Greene, et al.,“Protective Groups in Organic Synthesis”, John Wiley and Sons, SecondEdition, 1991, hereby incorporated by reference in its entirety.

As used herein, the term “alkyl” unless otherwise specified refers toboth branched and straight chain saturated aliphatic primary, secondary,and/or tertiary hydrocarbons of typically C₁ to C₂₁, for example C₁, C₂,C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, and specificallyincludes, but is not limited to, methyl, trifluoromethyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl,isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl,3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylhexyl,heptyl, octyl, nonyl, 3,7-dimethyloctyl, decyl, undecyl, dodecyl,tridecyl, 2-propylheptyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, and eicosyl.

The term “cycloalkyl” refers to cyclized alkyl groups. Exemplarycycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and adamantyl. Branchedcycloalkyl groups such as exemplary 1-methylcyclopropyl and2-methylcyclopropyl groups are included in the definition of cycloalkylas used in the present disclosure.

The term “arylalkyl”, as used herein, refers to a straight or branchedchain alkyl moiety having 1 to 8 carbon atoms that is substituted by anaryl group as defined herein, and includes, but is not limited to,benzyl, phenethyl, 2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl,2,4-dimethylbenzyl, 2-(4-ethylphenyl)ethyl, 3-(3-propylphenyl)propyl,and the like.

The term “aryl”, as used herein, and unless otherwise specified, refersto phenyl, biphenyl, naphthyl, anthracenyl, and the like.

The term “heteroaryl” refers to an aryl group where at least one carbonatom is replaced with a heteroatom (e.g. nitrogen, oxygen, sulfur) andcan be indolyl, furanyl, imidazolyl, triazolyl, triazinyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyrrolyl, pyrazinyl,tetrazolyl, pyridyl (or its N-oxide), thienyl, pyrimidinyl (or itsN-oxide), 1H-indolyl, isoquinolyl (or its N-oxide), or quinolyl (or itsN-oxide), for example.

The terms “alkoxy” and “alkyloxy” refer to a straight or branched alkylgroup attached to an oxygen atom. Exemplary alkyloxy groups include, butare not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, secondary butoxy, tertiary butoxy, pentoxy, isopentoxy,hexyloxy, heptyloxy, octyloxy, nonyloxy, and decyloxy.

The term “halogen”, as used herein, means fluoro, chloro, bromo andiodo.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample, and without limitation, isotopes of hydrogen include deuteriumand tritium, isotopes of carbon include ¹³C and ¹⁴C, isotopes ofnitrogen include ¹⁴N and ¹⁵N, and isotopes of oxygen include ¹⁶O, ¹⁷Oand ¹⁸O. Isotopically labeled compounds of the disclosure can generallybe prepared by conventional techniques known to those skilled in the artor by processes and methods analogous to those described herein, usingan appropriate isotopically labeled reagent in place of the non-labeledreagent otherwise employed.

According to a first aspect, the present disclosure relates to acompound of formula (I),

or a salt thereof, a solvate thereof, a tautomer thereof, or astereoisomer thereof.

R₁ is selected from the group consisting of an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, and an optionally substituted aryl.

In one embodiment, R₁ is an optionally substituted alkyl. In a preferredembodiment, R₁ is an unsubstituted alkyl, preferably a linear alkyl,preferably a linear C₁₋₆ alkyl, preferably a linear C₂₋₅ alkyl,preferably a linear C₃₋₄ alkyl. The carbon counts described hereinrefers to a number of carbon atoms of the alkyl group of R₁ whichexcludes the carbon atoms of optionally present substituents. Exemplarylinear alkyls include, but are not limited to methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Alternatively, R₁ isa branched alkyl, such as isopropyl, sec-butyl, isobutyl, isobutyl,tert-butyl, isopentyl, neopentyl, and isohexyl. In a most preferredembodiment, R₁ is methyl.

In another embodiment, R₁ is an optionally substituted aryl, such as anoptionally substituted phenyl, and naphthyl. Preferably R₁ is anoptionally substituted phenyl. The phenyl of R₁ may be substituted withat least one substituent such as an alkoxy (e.g. methoxy, ethoxy), analkyl, a halogen (e.g. chloro), and nitro. In a most preferredembodiment, R₁ is phenyl.

R₂ is an optionally substituted aryl, or an optionally substitutedheteroaryl.

In one embodiment, R₂ is an optionally substituted phenyl. The phenyl ofR₂ may be substituted with at least one substituent such as a hydroxy,an alkoxy (e.g. methoxy, ethoxy), an alkyl (e.g. methyl, ethyl), anamino (e.g. dimethylamino), halogen (e.g. fluoro, chloro, bromo), nitro,and cyano. In a preferred embodiment, R₂ is selected from the groupconsisting of phenyl, p-chlorophenyl, p-hydroxy-m-methoxyphenyl,p-methoxyphenyl. In a most preferred embodiment, R₂ is p-methoxyphenyl.

In another embodiment, R₂ is an optionally substituted heteroaryl.Exemplary applicable heteroaryls include, but are not limited to,2-furanyl, 2-thienyl, 3-methyl-2-furanyl, 3-methyl-2-thienyl,3-methyl-2-pyridinyl, and 4-methyl-2-pyridinyl. In a most preferredembodiment, R₂ is 2-furanyl.

R₃ is an optionally substituted aryl, or an optionally substitutedheteroaryl. In one embodiment, R₃ is an optionally substituted aryl,such as optionally substituted phenyl, naphthyl, and biphenyl. Inanother embodiment, R₃ is an optionally substituted heteroaryl, such as2-furanyl and 2-thienyl. In a preferred embodiment, R₃ is selected fromthe group consisting of phenyl, p-methoxyphenyl, 2-furanyl,p-fluorophenyl, and 4-1,1′-biphenyl. In a most preferred embodiment, R₃is phenyl.

R₄ is selected from the group consisting of a hydrogen, an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, and an optionally substituted aryl. In oneembodiment, R₄ is an unsubstituted alkyl, preferably a linear alkyl(e.g. methyl, ethyl, n-propyl). In another embodiment, R₄ is a branchedalkyl, such as isopropyl, and isobutyl. In a most preferred embodiment,R₄ is hydrogen.

R₅ and R₆ are independently selected from the group consisting of ahydrogen, an optionally substituted alkyl, an optionally substitutedcycloalkyl, and an optionally substituted arylalkyl. In one or moreembodiments, R₅ and R₆ are independently a hydrogen or an optionallysubstituted C₁₋₆ alkyl, C₂₋₅ alkyl, or a C₃₋₄ alkyl. In one embodiment,R₅ and R₆ are the same. In another embodiment, R₅ and R₆ are different.In a most preferred embodiment, R₅ and R₆ are hydrogen.

R₇, R₈, R₉, and R₁₀ are independently selected from the group consistingof a hydrogen, an optionally substituted alkyl (e.g. methyl, ethyl), anoptionally substituted cycloalkyl, an optionally substituted arylalkyl,a hydroxy, an alkoxy, and a halogen (e.g. chloro, bromo). In a preferredembodiment, R₇, R₈, R₉, and R₁₀ are hydrogen.

As used herein, the value of m denotes an alkyl chain of —CR₅R₆— groupsconnected between R₃ and amide group (—NR₄CO—) of the compound offormula (I). In one or more embodiments, m is an integer in a range of1-4, preferably 2-3. Preferably, m is 1 or 2. Most preferably, m is 1.

In some embodiments, the compound represented by formula (I) is one ormore of the following structures:

The double bond within the compound of formula (I) may have substituentsarranged in cis or trans configuration, preferably the cisconfiguration. For example, when R₂ is p-methoxyphenyl, it is understoodthat: the compound of formula (I) may be

The present disclosure further relates to a compound of formula (II),

or a salt thereof, a solvate thereof, a tautomer thereof, or astereoisomer thereof.

R₁′ is selected from the group consisting of an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, and an optionally substituted aryl.

In one embodiment, R₁′ is an optionally substituted alkyl. In apreferred embodiment, R₁′ is an unsubstituted alkyl, preferably a linearalkyl, preferably a linear C₁₋₆ alkyl, preferably a linear C₂₋₅ alkyl,preferably a linear C₃₋₄ alkyl. The carbon counts described hereinrefers to a number of carbon atoms of the alkyl group of R₁′ whichexcludes the carbon atoms of optionally present substituents. Exemplarylinear alkyls include, but are not limited to methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Alternatively, R₁′ isa branched alkyl, such as isopropyl, sec-butyl, isobutyl, isobutyl,tert-butyl, isopentyl, neopentyl, and isohexyl. In a most preferredembodiment, R₁′ is methyl.

In another embodiment, R₁′ is an optionally substituted aryl, such as anoptionally substituted phenyl, and naphthyl. Preferably R₁′ is anoptionally substituted phenyl. The phenyl of R₁′ may be substituted withat least one substituent such as an alkoxy (e.g. methoxy, ethoxy), analkyl, a halogen (e.g. chloro), and nitro. In a most preferredembodiment, R₁′ is phenyl.

R₂′ is an optionally substituted aryl, or an optionally substitutedheteroaryl.

In one embodiment, R₂′ is an optionally substituted phenyl. The phenylof R₂′ may be substituted with at least one substituent such as ahydroxy, an alkoxy (e.g. methoxy, ethoxy), an alkyl (e.g. methyl,ethyl), an amino (e.g. dimethylamino), a halogen (e.g. fluoro, chloro,bromo), nitro, and cyano. In a preferred embodiment, R₂′ is selectedfrom the group consisting of phenyl, p-chlorophenyl,p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, and p-fluoro-o-methylphenyl.In another embodiment, R₂′ is an optionally substituted heteroaryl.Exemplary applicable heteroaryls include, but are not limited to,2-furanyl, 2-thienyl, 3-methyl-2-furanyl, 3-methyl-2-thienyl,3-methyl-2-pyridinyl, and 4-methyl-2-pyridinyl. In a most preferredembodiment, R₂′ is p-chlorophenyl.

R₃′ is an optionally substituted aryl, or an optionally substitutedheteroaryl. In one embodiment, R₃′ is an optionally substituted aryl,such as optionally substituted phenyl, naphthyl, and biphenyl.Preferably, R₃′ is a phenyl that is optionally substituted by one ormore groups such as an alkyl, an alkoxy (e.g. methoxy, ethoxy), and ahalogen (e.g. fluoro, chloro, bromo). In another embodiment, R₃′ is anoptionally substituted heteroaryl. Exemplary applicable heteroarylsinclude, but are not limited to, 2-furanyl, 2-thienyl,3-methyl-2-furanyl, 3-methyl-2-thienyl, 3-methyl-2-pyridinyl, and4-methyl-2-pyridinyl. In a preferred embodiment, R₃′ is selected fromthe group consisting of phenyl, p-methoxyphenyl, 2-furanyl,p-fluorophenyl, and 4-1,1′-biphenyl. In a most preferred embodiment, R₃′is phenyl.

R₄′ is selected from the group consisting of a hydrogen, an optionallysubstituted alkyl, an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, and an optionally substituted aryl. In oneembodiment, R₄′ is an unsubstituted alkyl, preferably a linear alkyl(e.g. methyl, ethyl, n-propyl). In another embodiment, R₄′ is a branchedalkyl, such as isopropyl, and isobutyl. In a preferred embodiment, R₄′is hydrogen or methyl. Most preferably, R₄′ is hydrogen.

R₅′ and R₆′ are independently selected from the group consisting of ahydrogen, an optionally substituted alkyl, an optionally substitutedcycloalkyl, and an optionally substituted arylalkyl. In one or moreembodiments, R₅′ and R₆′ are independently a hydrogen or an optionallysubstituted C₁₋₆ alkyl, C₂₋₅ alkyl, or a C₃₋₄ alkyl. In one embodiment,R₅′ and R₆′ are the same. In another embodiment, R₅′ and R₆′ aredifferent. In a preferred embodiment, R₅′ is hydrogen, and R₆′ ishydrogen, or methyl. In a most preferred embodiment, R₅′ and R₆′ arehydrogen.

R₇′, R₈′, R₉′, and R₁₀′ are independently selected from the groupconsisting of a hydrogen, an optionally substituted alkyl (e.g. methyl,ethyl), an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, a hydroxy, an alkoxy, and a halogen (e.g. chloro, bromo). Ina preferred embodiment, R₇′, R₈′, R₉′, and R₁₀′ are hydrogen.

As used herein, the value of n denotes an alkyl chain of —CR₅′R₆′—groups connected between R₃′ and amide group (—NR₄′CO—) of the compoundof formula (II). In one or more embodiments, n is an integer in a rangeof 1-4, preferably 2-3. Preferably, n is 1 or 2. Most preferably, n is1.

In some embodiments, the compound represented by formula (II) is one ormore of the following structures:

The double bond within the compound of formula (I) may have substituentsarranged in cis or trans conformation, preferably the cis configuration.For example,

The double bond within the compound of formula (II) may havesubstituents arranged in cis or trans configuration, preferably the cisconfiguration. For example, when R₂′ is p-chlorophenyl, it is understoodthat: the compound of formula (II) may be

The present disclosure further relates to a compound of formula (III),

or a salt thereof, a solvate thereof, a tautomer thereof, or astereoisomer thereof.

R₁″ is selected from the group consisting of an optionally substitutedalkyl, an optionally substituted cycloalkyl, an optionally substitutedarylalkyl, and an optionally substituted aryl.

In one embodiment, R₁″ is an optionally substituted alkyl. In apreferred embodiment, R₁″ is an unsubstituted alkyl, preferably a linearalkyl, preferably a linear C₁₋₆ alkyl, preferably a linear C₂₋₅ alkyl,preferably a linear C₃₋₄ alkyl. The carbon counts described hereinrefers to a number of carbon atoms of the alkyl group of R₁″ whichexcludes the carbon atoms of optionally present substituents. Exemplarylinear alkyls include, but are not limited to methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Alternatively, R₁″ isa branched alkyl, such as isopropyl, sec-butyl, isobutyl, isobutyl,tert-butyl, isopentyl, neopentyl, and isohexyl. In a most preferredembodiment, R₁″ is methyl.

In another embodiment, R₁″ is an optionally substituted aryl, such as anoptionally substituted phenyl, and naphthyl. For example, R₁″ is anoptionally substituted phenyl. The phenyl of R₁″ may be substituted withat least one substituent such as an alkoxy (e.g. methoxy, ethoxy), analkyl, a halogen (e.g. chloro), and nitro. In a preferred embodiment,R₁″ is phenyl.

R₂″ is an optionally substituted aryl, or an optionally substitutedheteroaryl. In one embodiment, R₂″ is an optionally substituted phenyl.The phenyl of R₂″ may be optionally substituted with at least onesubstituent such as a hydroxy, an alkoxy (e.g. methoxy, ethoxy), analkyl (e.g. methyl, ethyl), an amino (e.g. dimethylamino), a halogen(e.g. fluoro, chloro, bromo), nitro, and cyano. Exemplary R₂″ includephenyl, p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, andp-fluoro-o-methylphenyl. In another embodiment, R₂″ is an optionallysubstituted heteroaryl. Exemplary applicable heteroaryls include, butare not limited to, 2-furanyl, 2-thienyl, 3-methyl-2-furanyl,3-methyl-2-thienyl, 3-methyl-2-pyridinyl, and 4-methyl-2-pyridinyl. In amost preferred embodiment, R₂″ is phenyl.

R₇″, R₈″, R₉″, R₁₀″, and R₁₁ are independently selected from the groupconsisting of a hydrogen, an optionally substituted alkyl (e.g. methyl,ethyl, n-propyl), an optionally substituted cycloalkyl, an optionallysubstituted arylalkyl, a hydroxy, an alkoxy, and a halogen (e.g. chloro,bromo). In a preferred embodiment, R₇″, R₈″, R₉″, and R₁₀″ are hydrogen.In another preferred embodiment, R₁₁ is methyl.

In one embodiment, the compound represented by formula (III) is

In a preferred embodiment, the compound of formula (III) has cisconfiguration.

In at least one embodiment, the compound is represented by formula (I),and R₁ is methyl, R₂ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, and p-methoxyphenyl,preferably p-methoxyphenyl, and R₃ is phenyl. In another embodiment, R₁is phenyl, R₂ is 2-furanyl, and R₃ is phenyl.

In at least one embodiment, the compound is represented by formula (II),and R₁′ is phenyl, R₂′ is p-chlorophenyl, and R₃′ is phenyl.

In a most preferred embodiment, the compound is selected from the groupconsisting of

The compounds of the present disclosure may be prepared by methods knownto those of ordinary skills in the art. The following methods set forthbelow are provided for illustrative purposes and are not intended tolimit the scope of the disclosure.

The compounds of formula (I) may, for example, be synthesized accordingto a process illustrated in FIG. 5, route (b) using an aromatic amine offormula (IV)

or a salt, solvate, tautomer or stereoisomer thereof, and an oxazolonederivative of formula (V)

or a salt, solvate, tautomer or stereoisomer thereof, wherein R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀ and m are as previously specified.

The aromatic amine of formula (IV) may be formed via (i) a reactionbetween 2-(4-nitrophenyl)acetic acid with a reagent such as oxalylchloride, thionyl chloride, phosphorus trichloride (POCl₃), andphosphorous pentachloride (POCl₅) to form 2-(4-nitrophenyl)acetylchloride, or via any other activated acyl chemistry known to those ofordinary skill (e.g., anhydride, acyl bromide, etc.); (ii) an amidationreaction between the acyl group of 2-(4-nitrophenyl)acetyl chloride andan amine of formula (VI)

or a salt, solvate, or stereoisomer thereof, in the presence of a base,to form a nitrophenyl amide of formula (VII)

or a salt, solvate, tautomer or stereoisomer thereof, wherein R₃, R₄,R₅, R₆, R₇, R₈, R₉, R₁₀ and m are as previously specified; (iii) areduction reaction of the nitrophenyl amide of formula (VII) using areducing reagent, thereby forming the amine of formula (IV)

or a salt, solvate, tautomer or stereoisomer thereof.

Reduction methods of nitro groups are generally known to those ofordinary skills in the art. Exemplary reducing methods and/or reagentsinclude, but are not limited to, tin(II) chloride, catalytichydrogenation over palladium-on-carbon, Raney nickel, sodium sulfide,iron metal in acetic acid, sodium borohydride, lithium borohydride, andBaker's yeast.

The oxazolone derivative of formula (V) may be prepared via Erlenmeyerazlactone synthesis using a carboxylic acid of formula (VIII)

or a salt, solvate, tautomer or stereoisomer thereof, and an aldehyde offormula (IX)

or a salt, solvate, tautomer or stereoisomer thereof, in the presence ofa dehydrating agent (e.g. acetic anhydride, carbodiimides such asN,N-dicyclohexylcarbodiimide), and a base (e.g. sodium acetate), whereinR₁ and R₂ are as previously specified. In certain embodiments, catalystsmay be used to facilitate and/or accelerate the Erlenmeyer synthesis.Exemplary catalysts include, but are not limited to, zinc oxide,alumina, antimony pentafluoride, ruthenium chloride, bismuth acetate,and dodecatungstophosphoric acid.

The Erlenmeyer azlactone synthesis may be performed at a temperature ina range of 30-120° C., 50-100° C., or about 80° C. in neat condition(i.e. solvent free) or in a solvent such as methylene chloride,chloroform, methanol, ethanol, isopropanol, dimethylformamide,tetrahydrofuran, benzene, xylene, ethyl acetate, diethyl ether,acetonitrile, dimethyl sulfoxide, nitrobenzene, and mixtures thereof.

Compounds represented by formula (I) may be obtained by reacting theaforementioned amine of formula (IV) and the oxazolone derivative offormula (V) in the presence of a base (see FIG. 5, route (b)). Thisreaction may involve an initial ring opening of the oxazolone derivativeof formula (V) under basic condition generating a carboxy intermediate,which is followed by an intermolecular cyclization between the carboxyintermediate and the amine of formula (IV), thereby forming the compoundof formula (I) with an imidazolinone core. Non-limiting examples of baseinclude pyridine, sodium hydroxide, potassium hydroxide, trimethylamine,triethylamine, diisopropylethylamine (DIPEA), triisopropylamine,dimethylaminopropylamine, N-methylmorpholine, N-methylpyrrolidine, and4-dimethylaminopyridine (DMAP). In a preferred embodiment, the base ispyridine.

The aforementioned reaction may be performed via heating at atemperature in a range of 50-200° C., 70-150° C., or about 100° C. for2-24 hours, 4-12 hours, or about 8 hours, preferably in neat conditionor in a solvent. In a preferred embodiment, a molar ratio of the amineof formula (IV) to the oxazolone derivative of formula (V) is in a rangeof 0.7:1 to 2:1, preferably 0.9:1 to 1.5:1, or about 1:1. In anotherpreferred embodiment, a molar ratio of the base (e.g. pyridine) to theoxazolone derivative of formula (V) is in a range of 5:1 to 75:1,preferably 10:1 to 60:1, preferably 25:1 to 40:1, or about 37:1. Thereaction forming the compounds of formula (I) may be conducted in inertgas (e.g. nitrogen, argon, helium). Also, in some embodiments, thereaction may not be conducted in inert gas, but in a vacuum.

The compounds of formula (II) may be synthesized according to a processillustrated in FIG. 5, route (c) using an aromatic amine of formula (X)

or a salt, solvate, tautomer or stereoisomer thereof, and an oxazolonederivative of formula (XII)

or a salt, solvate, tautomer or stereoisomer thereof, wherein R₁′, R₂′,R₃′, R₄′, R₅′, R₆′, R₇′, R₈′, R₉′, R₁₀′ and n are as previouslyspecified. The compounds of formula (III) may be synthesized in asimilar fashion using an aromatic amine of formula (XII)

or a salt, solvate, tautomer or stereoisomer thereof, and an oxazolonederivative of formula (XIII)

or a salt, solvate, tautomer or stereoisomer thereof, wherein R₁″, R₂″,R₇″, R₈″, R₉″, R₁₀″ and R₁₁ are as previously specified. The aromaticamine of formula (X) and the oxazolone derivatives of formulae (XI) and(XIII) used herein may be prepared via above described procedures forcompounds of formula (I).

The oxazolone derivatives ((XI), (XIII)) may undergo nucleophilic attackfrom the amines ((X), (XII)), thereby forming a ring opening product(i.e. compounds of formulae (II) and (III)). The reaction forming thecompounds of formulae (II) and (III) may be performed via heating at atemperature in a range of 40−180° C., 60-120° C., or about 80° C. for2-24 hours, 4-12 hours, or about 8 hours, preferably in a solvent or inneat condition. Exemplary solvent useful for the reaction includeacetonitrile, tetrahydrofuran, benzene, xylene, ethyl acetate, methylenechloride, chloroform, dimethylformamide, diethyl ether, dimethylsulfoxide, nitrobenzene, and mixtures thereof. In a preferredembodiment, the solvent is acetonitrile. The reaction forming thecompounds of formulae (II) and (III) may be conducted in inert gas (e.g.nitrogen, argon, helium). Also, in some embodiments, the reaction maynot be conducted in inert gas, but in a vacuum.

In at least one embodiment, the above described reaction for theformation of compounds of formulae (II) and (III) does not involveadditional base (e.g. pyridine).

A molar ratio of the amine of formula (X) to the oxazolone derivative offormula (XI), may be in a range of 0.7:1 to 2:1, preferably 0.9:1 to1.5:1, or about 1:1. A molar ratio of the amine of formula (XII) to theoxazolone derivative of formula (XIII) may be in a range of 0.7:1 to2:1, preferably 0.9:1 to 1.5:1, or about 1:1. Ina preferred embodiment,the oxazolone derivative of formula (XI) or (XIII) is present in aconcentration of 0.05-1 mol/L, preferably 0.1-0.8 mol/L, more preferably0.2-0.5 mol/L, or about 0.34 mol/L relative to a total volume of thesolvent (e.g. acetonitrile).

Due to steric hindrance, the aforementioned oxazolone derivatives offormulae (V), (XI), and (XIII) prepared using Erlenmeyer chemistry maybe predominantly cis isomer. The trans isomer of the oxazolonederivatives can be obtained via methods known to those of ordinary skillin the art. For example, cis to trans isomerization may proceed viaheating, and irradiation with UV and/or visible light.

The progress of the reactions may be monitored by methods known to thoseof ordinary skill in the art, such as thin layer chromatography, gaschromatography, nuclear magnetic resonance, infrared spectroscopy, andhigh pressure liquid chromatography combined with ultraviolet detectionor mass spectroscopy. The compounds of formula (I) may be isolated andpurified by methods known to those of ordinary skill in the art, such ascrystallization, filtration through a celite containing cartridge,evaporating the reaction mixture to dryness, aqueous work-up, extractionwith organic solvents, distillation, column chromatography, and highpressure liquid chromatography (HPLC) on normal phase or reversed phase.Preferred methods include column chromatography and recrystallization.

According to a second aspect, the present disclosure relates to apharmaceutical composition containing the presently disclosedcompound(s) and a pharmaceutically acceptable carrier and/or excipient.

As used herein, a “composition” or a “pharmaceutical composition” refersto a mixture of the active ingredient with other chemical components,such as pharmaceutically acceptable carriers and excipients. One purposeof a composition is to facilitate administration of the compounddisclosed herein in any of its embodiments to a subject. Pharmaceuticalcompositions of the present disclosure may be manufactured by processeswell known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Depending on theintended mode of administration (oral, parenteral, or topical), thecomposition can be in the form of solid, semi-solid or liquid dosageforms, such as tablets, suppositories, pills, capsules, powders,liquids, or suspensions, preferably in unit dosage form suitable forsingle administration of a precise dosage.

The term “active ingredient”, as used herein, refers to an ingredient inthe composition that is biologically active, for example, a compoundrepresented by formula (I), a salt thereof, a solvate thereof, atautomer thereof, a stereoisomer thereof, a compound represented byformula (II), a salt thereof, a solvate thereof, a tautomer thereof, astereoisomer thereof, a compound represented by formula (III), a saltthereof, a solvate thereof, a tautomer thereof, a stereoisomer thereof,or any mixtures of these compounds. In some embodiments, other activeingredients in addition to the compound of the current disclosure may beincorporated into a pharmaceutical composition.

In one or more embodiments, the compound of the pharmaceuticalcomposition is selected from the group consisting of

In one embodiment, the pharmaceutical composition comprises 0.1-90 wt %of the compound disclosed herein in any of its embodiments relative to atotal weight of the pharmaceutical composition. In preferredembodiments, the pharmaceutical composition comprises at least 0.01 wt%, at least 0.05 wt %, at least 0.1 wt %, at least 0.5 wt %, at least 5wt %, at least 10 wt %, at least 15 wt %, at least 20 wt %, at least 25wt %, at least 30 wt %, at least 35 wt %, at least 40 wt %, at least 45wt %, at least 50 wt %, at least 55 wt %, at least 60 wt %, at least 65wt %, at least 70 wt %, at least 75 wt %, at least 80 wt %, at least 85wt %, at least 90 wt %, at least 95 wt %, at least 99 wt %, or at least99.9 wt % of the compound relative to a total weight of thepharmaceutical composition. The pharmaceutical composition may contain0.5-500 μM of the compound relative to a total volume of thecomposition, preferably 1-400 μM, preferably 10-300 μM, preferably20-200 μM of the compound relative to the total volume of thecomposition. In some embodiments, the composition comprises up to 0.1 wt%, up to 1 wt %, up to 5 wt %, up to 10 wt %, up to 25 wt %, or up to 50wt % of a pharmaceutically acceptable salt of the compound. In someembodiments, the composition comprises up to 0.1 wt %, 1 wt %, 5 wt %,or 10 wt % of a pharmaceutically acceptable solvate of the compound. Inone or more embodiments, the pharmaceutical composition comprises up to0.01%, up to 0.1%, up to 1%, up to 5%, or up to 10% by weight of thepharmaceutically acceptable carrier and/or excipient relative to a totalweight of the pharmaceutical composition. Preferably, the compositionmay further comprise pharmaceutically acceptable binders, such assucrose, lactose, xylitol, and pharmaceutically acceptable excipientssuch as calcium carbonate, calcium phosphate, and dimethyl sulfoxide(DMSO).

KX-01 and KX-02 developed by Athenex possess intriguing biologicalmechanisms against cancer cells. In addition to Src inhibition, thesetwo drugs also prevent cancer cell division via interference withtubulin [Tu, C.; Li, J.; Bu, Y.; Hangauer, D.; Qu, J., Anion-current-based, comprehensive and reproducible proteomic strategy forcomparative characterization of the cellular responses to novelanti-cancer agents in a prostate cell model. Journal of proteomics 2012,77, 187-201; and Anbalagan, M.; Ali, A.; Jones, R. K.; Marsden, C. G.;Sheng, M.; Carrier, L.; Bu, Y.; Hangauer, D.; Rowan, B. G.,Peptidomimetic Src/pretubulin inhibitor KX-01 alone and in combinationwith paclitaxel suppresses growth, metastasis in humanER/PR/HER2-negative tumor xenografts. Molecular cancer therapeutics2012, 11 (9), 1936-1947, each incorporated herein by reference in theirentirety].

It was found that the biphenylmethylcarboxamide pharmacophore of KXcompounds binds to substrate site in Src, but does not enter thejuxtaposed ATP pocket. Another mechanism of cytotoxic efficacy of KXcompounds involves their inhibition of tubulin. For example, KX2-391(FIG. 1) is a highly potent anticancer agent for the treatment ofseveral malignant tumors that has passed phase-III clinical trialsagainst actinic keratosis.

Previously, compounds having p-acylamino-N-benzylphenylacetamidepharmacophore were developed as anticancer agents using scaffold hoppingdesign technique (FIG. 1). This pharmacophore demonstratedanti-proliferative activity against cancer cell lines via dualinhibition of Src kinase and tubulin. For example, compounds KAC-03 andKAC-12 exhibited cytotoxic activities against various cancer cell lines(FIG. 1). Further structural modification of this pharmacophore mayenhance inhibition of kinases and/or tubulin and provide greateranticancer effect.

In some embodiments, the active ingredient of the current disclosure,e.g. a compound represented by formula (I), a salt thereof, a solvatethereof, a tautomer thereof, a stereoisomer thereof, a compoundrepresented by formula (II), a salt thereof, a solvate thereof, atautomer thereof, a stereoisomer thereof, a compound represented byformula (III), a salt thereof, a solvate thereof, a tautomer thereof, astereoisomer thereof, or any mixtures of these compounds, providesutility as an anticancer agent in reducing the viability of cancer cellsderived from human cancer cell lines including, but not limited to,breast cancer cell lines (e.g. MCF7, SK-BR-3), prostate cancer celllines (e.g. PC3), leukemia cell lines (e.g. HL-60), stomach cancer celllines (e.g. N87, SNU-16), colon cancer cell lines (e.g. HCT-116, HT-29),liver cancer cell lines (e.g. HepG2), lung cancer cell lines (e.g. A549,NCI-H460), brain tumor cell lines (e.g. U251), ovarian cancer cell lines(e.g. NCI-ADR/RES, OVCAR-03), renal cancer cell lines (e.g. 786-0), andmelanoma cell lines (e.g. UACC-62).

As used herein, other non-cancerous proliferative disorders that mayalso be treated by the currently disclosed pharmaceutical compositioninclude, without limitation, atherosclerosis, rheumatoid arthritis,psoriasis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of theliver, lymphoproliferative disorder, other disorders characterized byepidermal cell proliferation such as verruca (warts), and dermatitis.The active ingredient of the current disclosure may also exhibit othertherapeutic activities such as antimicrobial (e.g. antibacterial,antifungal, antiviral, antimycobacterial), antimalarial, pesticidal,antioxidant, as well as anti-inflammatory efficacies.

In some embodiments, the ability of the active ingredient to reduce theviability of cancer cells may be determined by contacting thepharmaceutical composition with the cancer cells and then performingcell viability assays. Methods of such assays include, but are notlimited to, ATP test, Calcein AM assay, clonogenic assay, ethidiumhomodimer assay, Evans blue assay, fluorescein diacetatehydrolysis/Propidium iodide staining assay, flow cytometry,Formazan-based assays (MTT, XTT), green fluorescent protein assay,lactate dehydrogenase (LDH) assay, sulforhodamine B (SRB) assay, methylviolet assay, propidium iodide assay, Resazurin assay, trypan blueassay, and TUNEL assay. In a preferred embodiment, a SRB assay is used.In another preferred embodiment, a Resazurin assay is used.

In some embodiments, the cancer cells are derived from human cancer celllines, including, but not limited to, breast cancer cell lines, e.g.,MDA-MB-231, MCF7, T47D, and VP303, prostate cancer cell lines, e.g.,PC3, VCaP, C4-2B, and MDA PCa 2b, leukemia cell lines, e.g., HL-60,CESS, CCRF-CEM, CEM/C1, KASUMI-1, ARH-77, stomach cancer cell lines,e.g., N87, SNU-16, SNU-5, SNU-1, KATO III, AGS, colon cancer cell lines,e.g., HCT15, MDST8, GP5d, HCT116, DLD1, HT29, SW620, SW403 and T84,liver cancer cell lines, e.g. HepG2, PLC/PRF/5, THLE-3, C3A, SNU-182,SNU-398, SNU-387, SNU-423, SNU-475, SNU-449, and Hep 3B2.1-7, lungcancer cell lines, e.g., A549, SHP-77, COR-L23/R, and NCI-H69/LX20,cervical cancer cell Lines, e.g., HeLa DH, HtTA-1, HRS, and C-4I,ovarian cancer cell lines, e.g., A2780, A2780cis, OV7, and PEO23, andskin cancer cell lines, e.g., C32TG, A375, and MCC26. In otherembodiments, the cancer cells are collected from a human patient who isat risk of having, is suspected of having, has been diagnosed with, oris being monitored for recurrence of at least one type of cancer,preferably breast cancer, prostate cancer, and/or leukemia.

As used herein, the term “cytotoxic effective amount” refers to aconcentration of the active ingredient that reduces the viability of thecancer cells by at least 5%, at least 10%, at least 15%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80% or at least 90%, relative to cancer cells not treated with theactive ingredient. The reduction in viability may occur no more than 10days, no more than 7 days, no more than 5 days, no more than 3 days, orno more than 2 days after the active ingredient is contacted with thecancer cells. In one embodiment, the cytotoxic effective amount may bethe IC₅₀ which is a concentration of the active ingredient which causesthe death of 50% of cancer cells in 12-72 hours, 20-48 hours, or about24 hours (1 day).

In one embodiment, the IC₅₀ of the presently disclosed compounds againstbreast cancer cells (e.g. MCF7) is in a range of 0.1-100 μM, preferably1-50 μM, more preferably 5-20 μM. In a preferred embodiment, thecompound is

or a mixture thereof, and the IC₅₀ against breast cancer cells is in arange of 0.1-5 μM, preferably 0.15-3 μM, more preferably 0.5-1 μM.

In another embodiment, the IC₅₀ of the presently disclosed compoundsagainst prostate cancer cells (e.g. PC3) is in a range of 0.2-100 μM,preferably 1-50 μM, more preferably 5-20 μM. In a preferred embodiment,the compound is

or a mixture thereof, and the IC₅₀ against prostate cancer cells is in arange of 0.2-7 μM, preferably 0.3-5 μM, more preferably 0.5-2 μM.

In another embodiment, the IC₅₀ of the presently disclosed compoundsagainst leukemia cells is in a range of 100-5,000 nM, preferably200-1,000 nM, more preferably 400-600 nM. In a preferred embodiment, thecompound is

and the IC₅₀ against leukemia cells is in a range of 100-400 nM,preferably 200-300 nM, or about 260 nM.

In some embodiments, other active ingredients in addition to thecompound(s) of the current disclosure may be incorporated into thepharmaceutical composition. In one embodiment, the pharmaceuticalcomposition includes a second active ingredient that is chemicallydistinct from the compounds of formulae (I), (II), and (III), such as achemotherapeutic agent or an anticancer agent, for the treatment orprevention of neoplasm, of tumor or cancer cell division, growth,proliferation and/or metastasis in the subject; induction of death orapoptosis of tumor and/or cancer cells; and/or any other forms ofproliferative disorder.

The anticancer agent is at least one of a mitotic inhibitor; analkylating agent; an antimetabolite; a cell cycle inhibitor; an enzyme;a topoisomerase inhibitor; a biological response modifier; ananti-hormone; a tubulin inhibitor; a tyrosine-kinase inhibitor; anantiangiogenic agent such as MMP-2, MMP-9 and COX-2 inhibitor; ananti-androgen; a platinum coordination complex (oxaliplatin,carboplatin); a substituted urea such as hydroxyurea; a methylhydrazinederivative; an adrenocortical suppressant, e.g., mitotane,aminoglutethimide; a hormone and/or hormone antagonist such as theadrenocorticosteriods (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate), an estrogen (e.g., diethylstilbestrol);an antiestrogen such as tamoxifen; androgen, e.g., testosteronepropionate; and an aromatase inhibitor, such as anastrozole, andAROMASIN (exemestane).

Exemplary anticancer agents include, but are not limited to, tubulinbinding agents including paclitaxel, epothilone, docetaxel,discodermolide, etoposide, vinblastine, vincristine, teniposide,vinorelbine, and vindesine; tyrosine-kinase inhibitors includingimatinib, nilotinib, dasatinib, bosutinib, ponatinib, and bafetinib;alkylating antineoplastic agents including busulfan, carmustine,chlorambucil, cyclophosphamide, cyclophosphamide, dacarbazine,ifosfamide, lomustine, mechlorethamine, melphalan, mercaptopurine,procarbazine; antimetabolites including cladribine, cytarabine,fludarabine, gemcitabine, pentostatin, 5-fluorouracil, clofarabine,capecitabine, methotrexate, thioguanine; cytotoxic antibiotics includingdaunorubicin, doxorubicin, idarubicin, mitomycin, actinomycin,epirubicin; topoisomerase inhibitors including irinotecan, mitoxantrone,topotecan, and mixtures thereof.

As used herein, a “pharmaceutically acceptable carrier” refers to acarrier or diluent that does not cause significant irritation to anorganism, does not abrogate the biological activity and properties ofthe administered active ingredient, and/or does not interact in adeleterious manner with the other components of the composition in whichit is contained. The term “carrier” encompasses any excipient, binder,diluent, filler, salt, buffer, solubilizer, lipid, stabilizer, or othermaterial well known in the art for use in pharmaceutical formulations.The choice of a carrier for use in a composition will depend upon theintended route of administration for the composition. The preparation ofpharmaceutically acceptable carriers and formulations containing thesematerials is described in, e.g. Remington's Pharmaceutical Sciences,21st Edition, ed. University of the Sciences in Philadelphia,Lippincott, Williams & Wilkins, Philadelphia Pa., 2005, which isincorporated herein by reference in its entirety). Examples ofphysiologically acceptable carriers include antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN® (ICI, Inc.;Bridgewater, N.J.), polyethylene glycol (PEG), and PLURONICS™ (BASF;Florham Park, N.J.). An “excipient” refers to an inert substance addedto a composition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils, and polyethylene glycols.

In one or more embodiments, the pharmaceutically acceptable carrierand/or excipient is at least one selected from the group consisting of abuffer, an inorganic salt, a fatty acid, a vegetable oil, a syntheticfatty ester, a surfactant, and a polymer.

Exemplary buffers include, but are not limited to, phosphate buffers,citrate buffer, acetate buffers, borate buffers, carbonate buffers,bicarbonate buffers, and buffers with other organic acids and salts.

Exemplary inorganic salts include, but are not limited to, calciumcarbonate, calcium phosphate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc oxide, zinc sulfate, andmagnesium trisilicate.

Exemplary fatty acids include, but are not limited to, an omega-3 fattyacid (e.g., linolenic acid, docosahexaenoic acid, eicosapentaenoic acid)and an omega-6 fatty acid (e.g., linoleic acid, eicosadienoic acid,arachidonic acid). Other fatty acids, such as oleic acid, palmitoleicacid, palmitic acid, stearic acid, and myristic acid, may be included.

Exemplary vegetable oils include, but are not limited to, avocado oil,olive oil, palm oil, coconut oil, rapeseed oil, soybean oil, corn oil,sunflower oil, cottonseed oil, and peanut oil, grape seed oil, hazelnutoil, linseed oil, rice bran oil, safflower oil, sesame oil, brazil nutoil, carapa oil, passion fruit oil, and cocoa butter.

Exemplary synthetic fatty esters include, without limitation, methyl,ethyl, isopropyl and butyl esters of fatty acids (e.g., isopropylpalmitate, glyceryl stearate, ethyl oleate, isopropyl myristate,isopropyl isostearate, diisopropyl sebacate, ethyl stearate, di-n-butyladipate, dipropylene glycol pelargonate), C₁₂-C₁₆ fatty alcohol lactates(e.g., cetyl lactate and lauryl lactate), propylene dipelargonate,2-ethylhexyl isononoate, 2-ethylhexyl stearate, isopropyl lanolate,2-ethylhexyl salicylate, cetyl myristate, oleyl myristate, oleylstearate, oleyl oleate, hexyl laurate, isohexyl laurate, propyleneglycol fatty ester, and polyoxyethylene sorbitan fatty ester. As usedherein, the term “propylene glycol fatty ester” refers to a monoether ordiester, or mixtures thereof, formed between propylene glycol orpolypropylene glycol and a fatty acid. The term “polyoxyethylenesorbitan fatty ester” denotes oleate esters of sorbitol and itsanhydrides, typically copolymerized with ethylene oxide.

Surfactants may act as detergents, wetting agents, emulsifiers, foamingagents, and dispersants. Surfactants that may be present in thecompositions of the present disclosure include zwitterionic (amphoteric)surfactants, e.g., phosphatidylcholine, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),anionic surfactants, e.g., sodium lauryl sulfate, sodium octanesulfonate, sodium decane sulfonate, and sodium dodecane sulfonate,non-ionic surfactants, e.g., sorbitan monolaurate, sorbitanmonopalmitate, sorbitan trioleate, polysorbates such as polysorbate 20(Tween 20), polysorbate 60 (Tween 60), and polysorbate 80 (Tween 80),cationic surfactants, e.g., decyltrimethylammonium bromide,dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide,tetradecyltrimethyl-ammonium chloride, and dodecylammonium chloride, andcombinations thereof.

Exemplary polymers include, without limitation, polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyurethanes,polyesteramides, polyorthoesters, polydioxanones, polyacetals,polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes,polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), poly(maleic anhydride), apolyvinyl alcohols, and copolymers, terpolymers, or combinations ormixtures therein. The copolymer/terpolymer may be a randomcopolymer/terpolymer, or a block copolymer/terpolymer.

Depending on the route of administration e.g. oral, parental, ortopical, the pharmaceutical composition may be in the form of soliddosage form such as tablets, caplets, capsules, powders, and granules,semi-solid dosage form such as ointments, creams, lotions, gels, pastes,and suppositories, liquid dosage forms such as solutions, anddispersions, inhalation dosage form such as aerosols, and spray, ortransdermal dosage form such as patches.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive ingredient is ordinarily combined with one or more adjuvantsappropriate to the indicated route of administration. If administeredper os, the active ingredient can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering ingredients such as sodium citrate,magnesium or calcium carbonate or bicarbonate. Tablets and pills canadditionally be prepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting ingredients,emulsifying and suspending ingredients, and sweetening, flavouring, andperfuming ingredients.

For therapeutic purposes, formulations for parenteral administration canbe in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. The term “parenteral”, as used herein,includes intravenous, intravesical, intraperitoneal, subcutaneous,intramuscular, intralesional, intracranial, intrapulmonal, intracardial,intrasternal, and sublingual injections, or infusion techniques. Thesesolutions and suspensions can be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The active ingredientcan be dissolved in water, polyethylene glycol, propylene glycol,ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzylalcohol, sodium chloride, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting ingredients and suspendingingredients. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a non-toxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or di-glycerides. In addition, fatty acids,such as oleic acid, find use in the preparation of injectables. Dimethylacetamide, surfactants including ionic and non-ionic detergents,polyethylene glycols can be used. Mixtures of solvents and wettingingredients such as those discussed above are also useful.

Suppositories for rectal administration can be prepared by mixing theactive ingredient with a suitable non-irritating excipient, such ascocoa butter, synthetic mono-, di-, or triglycerides, fatty acids, andpolyethylene glycols that are solid at ordinary temperatures but liquidat the rectal temperature and will therefore melt in the rectum andrelease the drug.

Topical administration may involve the use of transdermal administrationsuch as transdermal patches or iontophoresis devices. Formulation ofdrugs is discussed in, for example, Hoover, J. E. Remington'spharmaceutical sciences, Mack Publishing Co., Easton, Pa., 1975; andLiberman, H. A.; Lachman, L., Eds. Pharmaceutical dosage forms, MarcelDecker, New York, N.Y., 1980, which are incorporated herein by referencein their entirety.

In other embodiments, the pharmaceutical composition having thepresently disclosed compound(s), the salt thereof, the solvate thereof,the tautomer thereof, the stereoisomer thereof, or the mixture thereofhas different release rates categorized as immediate release andcontrolled- or sustained-release.

As used herein, immediate release refers to the release of an activeingredient substantially immediately upon administration. In anotherembodiment, immediate release occurs when there is dissolution of anactive ingredient within 1-20 minutes after administration. Dissolutioncan be of all or less than all (e.g. about 70%, about 75%, about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, 99.9%, or99.99%) of the active ingredient. In another embodiment, immediaterelease results in complete or less than complete dissolution withinabout 1 hour following administration. Dissolution can be in a subject'sstomach and/or intestine. In one embodiment, immediate release resultsin dissolution of an active ingredient within 1-20 minutes afterentering the stomach. For example, dissolution of 100% of an activeingredient can occur in the prescribed time. In another embodiment,immediate release results in complete or less than complete dissolutionwithin about 1 hour following rectal administration. In someembodiments, immediate release is through inhalation, such thatdissolution occurs in a subject's lungs.

Controlled-release, or sustained-release, refers to a release of anactive ingredient from a composition or dosage form in which the activeingredient is released over an extended period of time. In oneembodiment, controlled-release results in dissolution of an activeingredient within 20-180 minutes after entering the stomach. In anotherembodiment, controlled-release occurs when there is dissolution of anactive ingredient within 20-180 minutes after being swallowed. Inanother embodiment, controlled-release occurs when there is dissolutionof an active ingredient within 20-180 minutes after entering theintestine. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour followingadministration. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour following oraladministration. In another embodiment, controlled-release results insubstantially complete dissolution after at least 1 hour followingrectal administration. In one embodiment, the pharmaceutical compositiondescribed herein is not a controlled-release composition.

According to a third aspect, the present disclosure relates to a methodfor treating a proliferative disorder. The method involves administeringthe pharmaceutical composition of the second aspect to a subject in needof therapy.

In one or more embodiments, the proliferative disorder is cancer. Insome embodiments, the disclosed method of the current aspect is fortreating cancer of the blood, stomach, breast, colon, brain, bladder,lung, cervix, ovary, rectum, pancreas, skin, prostate gland, spleen,liver, kidney, head, neck, testicle, bone, bone marrow, thyroid gland,or central nervous system. In a preferred embodiment, the cancer is atleast one selected from the group consisting of breast cancer, prostatecancer, and leukemia.

As used herein, the terms “treat”, “treatment”, and “treating” in thecontext of the administration of a therapy to a subject in need thereofrefer to the reduction or inhibition of the progression and/or durationof a disease (e.g. cancer), the reduction or amelioration of theseverity of the disease, and/or the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies.“Treating” or “treatment” of the disease includes preventing the diseasefrom occurring in a subject that may be predisposed to the disease butdoes not yet experience or exhibit symptoms of the disease (prophylactictreatment), inhibiting the disease (slowing or arresting itsdevelopment), ameliorating the disease, providing relief from thesymptoms or side-effects of the disease (including palliativetreatment), and relieving the disease (causing regression of thedisease). With regard to the disease, these terms simply mean that oneor more of the symptoms of the disease will be reduced. Such terms mayrefer to one, two, three, or more results following the administrationof one, two, three, or more therapies: (1) a stabilization, reduction(e.g. by more than 10%, 20%, 30%, 40%, 50%, preferably by more than 60%of the population of cancer cells and/or tumor size beforeadministration), or elimination of the cancer cells, (2) inhibitingcancerous cell division and/or cancerous cell proliferation, (3)relieving to some extent (or, preferably, eliminating) one or moresymptoms associated with a pathology related to or caused in part byunregulated or aberrant cellular division, (4) an increase indisease-free, relapse-free, progression-free, and/or overall survival,duration, or rate, (5) a decrease in hospitalization rate, (6) adecrease in hospitalization length, (7) eradication, removal, or controlof primary, regional and/or metastatic cancer, (8) a stabilization orreduction (e.g. by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,preferably at least 80% relative to the initial growth rate) in thegrowth of a tumor or neoplasm, (9) an impairment in the formation of atumor, (10) a reduction in mortality, (11) an increase in the responserate, the durability of response, or number of patients who respond orare in remission, (12) the size of the tumor is maintained and does notincrease or increases by less than 10%, preferably less than 5%,preferably less than 4%, preferably less than 2%, (13) a decrease in theneed for surgery (e.g. colectomy, mastectomy), and (14) preventing orreducing (e.g. by more than 10%, more than 30%, preferably by more than60% of the population of metastasized cancer cells beforeadministration) the metastasis of cancer cells.

The term “subject” and “patient” are used interchangeably. As usedherein, they refer to any subject for whom or which therapy, includingwith the compositions according to the present disclosure is desired. Inmost embodiments, the subject is a mammal, including but not limited toa human, a non-human primate such as a chimpanzee, a domestic livestocksuch as a cattle, a horse, a swine, a pet animal such as a dog, a cat,and a rabbit, and a laboratory subject such as a rodent, e.g. a rat, amouse, and a guinea pig. In preferred embodiments, the subject is ahuman.

As used herein, a subject in need of therapy includes a subject alreadywith the disease, a subject which does not yet experience or exhibitsymptoms of the disease, and a subject predisposed to the disease. Inpreferred embodiments, the subject is a person who is predisposed tocancer, e.g. a person with a family history of cancer. Women who have(i) certain inherited genes (e.g. mutated BRCA1 and/or mutated BRCA2),(ii) been taking estrogen alone (without progesterone) after menopausefor many years (at least 5, at least 7, or at least 10), and/or (iii)been taking fertility drug clomiphene citrate, are at a higher risk ofcontracting breast cancer. People who (i) have certain inherited mutatedgenes (e.g. mutated RNASEL, mutated BRCA1 and/or mutated BRCA2), (ii)had inflammation in the prostate, and/or (iii) are obese are at a higherrisk of contracting prostate cancer. People who (i) had chemotherapy andradiation therapy for other cancers, (ii) has genetic disorders, such asDown syndrome, and/or (iii) exposure to certain chemicals, such asbenzene are at a higher risk of contracting leukemia.

In another embodiment, the subject refers to a cancer patient who hasbeen previously treated and/or administered with a tyrosine-kinaseinhibitor such as imatinib, nilotinib, dasatinib, bosutinib, ponatinib,and bafetinib, and developed drug resistance via (i) Bcr-Abl dependentmechanisms involving Bcr-Abl duplication, Bcr-Abl mutation, T3151mutation, and/or P-loop mutations, or (ii) Bcr-Abl Independentmechanisms involving drug efflux caused by P-glycoproteins, drug importby organic cation transporter 1, and/or alternative signaling pathwayactivation.

In another embodiment, the subject refers to a cancer patient who hasbeen previously administered and/or treated with a tubulin binding drugsuch as paclitaxel, epothilone, docetaxel, discodermolide, etoposide,vinblastine, vincristine, teniposide, vinorelbine, and vindesine, anddeveloped resistance to the tubulin binding drug.

In at least one embodiment, the subject has leukemia, prostate, and/orbreast cancer and is currently undergoing, or has completed atyrosine-kinase inhibitor based and/or tubulin inhibitor basedchemotherapy regimen.

The terms “administer”, “administering”, “administration”, and the like,as used herein, refer to the methods that may be used to enable deliveryof the active ingredient and/or the composition to the desired site ofbiological action. Routes or modes of administration are as set forthherein. These methods include, but are not limited to, oral routes,intraduodenal routes, parenteral injection (including intravenous,subcutaneous, intraperitoneal, intramuscular, intravascular, orinfusion), topical and rectal administration. Those of ordinary skill inthe art are familiar with administration techniques that can be employedwith the complexes and methods described herein. In a preferredembodiment, the active ingredient and/or the pharmaceutical compositiondescribed herein are administered orally.

In one embodiment, the pharmaceutical composition administered comprisesthe compound of formula (I), or a salt thereof, a solvate thereof, atautomer thereof, or a stereoisomer thereof, in which R₁ is methyl, orphenyl, R₂ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, and2-furanyl, and R₃ is phenyl. In a most preferred embodiment, thepharmaceutical composition administered comprises a compound of formula(I) which is selected from the group consisting of

In another embodiment, the pharmaceutical composition administeredcomprises the compound of formula (II), or a salt thereof, a solvatethereof, a tautomer thereof, or a stereoisomer thereof, in which R₁′ ismethyl, or phenyl, R₂′ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, andp-fluoro-o-methylphenyl, and R₃′ is selected from the group consistingof phenyl, p-methoxyphenyl, 2-furanyl, p-fluorophenyl, and4-1,1′-biphenyl. In a most preferred embodiment, the pharmaceuticalcomposition administered comprises a compound of formula (II) which is

The dosage amount and treatment duration are dependent on factors, suchas bioavailability of a drug, administration mode, toxicity of a drug,gender, age, lifestyle, body weight, the use of other drugs and dietarysupplements, the disease stage, tolerance and resistance of the body tothe administered drug, etc., and then determined and adjustedaccordingly. The terms “effective amount”, “therapeutically effectiveamount”, or “pharmaceutically effective amount” refer to that amount ofthe active ingredient being administered which will relieve to someextent one or more of the symptoms of the disease being treated. Theresult can be a reduction and/or alleviation of the signs, symptoms, orcauses of a disease, or any other desired alteration of a biologicalsystem. An appropriate “effective amount” may differ from one individualto another. An appropriate “effective amount” in any individual case maybe determined using techniques, such as a dose escalation study. In oneor more embodiments, an effective amount of the compound disclosedherein in a range of 0.1-500 mg/kg, preferably 1-200 mg/kg, morepreferably 10-50 mg/kg is administered per body weight of the subject.However, in certain embodiments, the effective amount of the compound isless than 0.1 mg/kg or greater than 500 mg/kg.

In treating certain cancers, the best approach is often a combination ofsurgery, radiotherapy, and/or chemotherapy. Therefore, in at least oneembodiment, the pharmaceutical composition is employed in conjunctionwith radiotherapy. In another embodiment, the pharmaceutical compositionis employed with surgery. The radiotherapy and/or surgery may beperformed before or after the pharmaceutical composition isadministered.

A treatment method may comprise administering a pharmaceuticalcomposition containing the compound of the current disclosure in any ofits embodiments as a single dose or multiple individual divided doses.In some embodiments, the composition is administered at various dosages(e.g. a first dose with an effective amount of 200 mg/kg and a seconddose with an effective amount of 50 mg/kg). In some embodiments, theinterval of time between the administration of the composition and theadministration of one or more additional therapies may be about 1-5minutes, 1-30 minutes, 30 minutes to 60 minutes, 1 hour, 1-2 hours, 2-6hours, 2-12 hours, 12-24 hours, 1-2 days, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 26weeks, 52 weeks, 11-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks,40-50 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months,7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2years, or any period of time in between. Preferably, the composition isadministered once daily for at least 2 days, at least 5 days, at least 6days, or at least 7 days. In certain embodiments, the composition andone or more additional therapies are administered less than 1 day, lessthan 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks,less than 1 month, less than 2 months, less than 3 months, less than 6months, less than 1 year, less than 2 years, or less than 5 years apart.

The methods for treating cancer and other proliferative disordersdescribed herein inhibit, remove, eradicate, reduce, regress, diminish,arrest or stabilize a cancerous tumor, including at least one of thetumor growth, tumor cell viability, tumor cell division andproliferation, tumor metabolism, blood flow to the tumor and metastasisof the tumor. In some embodiments, the size of a tumor, whether byvolume, weight or diameter, is reduced after the treatment by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100%, relative to the tumor size before treatment. In otherembodiments, the size of a tumor after treatment does not reduce but ismaintained the same as the tumor size before treatment. Methods ofassessing tumor size include, but are not limited to, CT scan, MRI,DCE-MRI and PET scan.

In one embodiment, the method disclosed herein may reduce the number ofabnormal peripheral blood mononuclear cells in a leukemia patient, whomay be afflicted with acute lymphoblastic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloidleukemia (CML). Preferably, the number of abnormal peripheral bloodmononuclear cells is reduced after the treatment by at least 5%, atleast 10%, at least 20%, at least 30%, or at least 40%, and up to 100%,up to 99%, up to 95%, up to 90%, up to 80%, or up to 60%, relative to aninitial number of abnormal peripheral blood mononuclear cells beforetreatment.

In most embodiments, the method further comprises measuring aconcentration of a biomarker and/or detecting a mutation in a biomarkerbefore and/or after the pharmaceutical composition comprising thecompound of the present disclosure is administered. As used herein, theterm “biomarker” refers to a characteristic that is objectively measuredand evaluated as an indicator of normal biological processes, pathogenicprocesses or pharmacological responses to a therapeutic intervention.Generic cancer biomarkers include circulating tumor DNA (ctDNA) andcirculating tumor cells (CTC). Exemplary biomarkers for breast cancerinclude, without limitation, BRCA1, BRCA2, HER-2, estrogen receptor,progesterone receptor, cancer antigen 15-3, cancer antigen 27.29,carcinoembryonic antigen, Ki67, cyclin D1, cyclin E, and ERβ. Exemplarybiomarkers for prostate cancer include, without limitation, tPSA, fPSA,p2PSA, HOXC6 DLX1, GSTP1, RASSF1, and APC. In one embodiment, leukemiapatient's response to the treatment may be monitored by (i) measuringthe complete blood count, (ii) observing the disappearance/reduction inoccurrences of abnormal cytogenetic markers detected at the time ofdiagnosis, and/or (iii) observing the disappearance/reduction inoccurrences of BCR/ABL mutational copies detected at the time ofdiagnosis.

The mutation in the biomarker may be detected by procedures such asrestriction fragment length polymorphism (RFLP), polymerase chainreaction (PCR) assay, multiplex ligation-dependent probe amplification(MLPA), denaturing gradient gel electrophoresis (DGGE), single-strandconformation polymorphism (SSCP), hetero-duplex analysis, proteintruncation test (PTT), and oligonucleotide ligation assay (OLA). Theprocedures to detect the mutation are well-known to those of ordinaryskill in the art.

The term “sample” used herein refers to any biological sample obtainedfrom the subject in need of therapy including a single cell, multiplecells, fragments of cells, a tissue sample, and/or body fluid.Specifically, the biological sample may include red blood cells, whiteblood cells, platelets, hepatocytes, epithelial cells, endothelialcells, a skin biopsy, a mucosa biopsy, an aliquot of urine, saliva,whole blood, serum, plasma, lymph. In some embodiments, the biologicalsample is taken from a tumor.

The concentration level of the cancer biomarker in a sample may bemeasured by an assay, for example an immunoassay. Typical immunoassaymethods include, without limitation, enzyme-linked immunosorbent assay(ELISA), enzyme-linked immunospot assay (ELISPOT), Western blotting,immunohistochemistry (IHC), immunocytochemistry, immunostaining, andmultiple reaction monitoring (MRM) based mass spectrometric immunoassay.The protocol for measuring the concentration of the biomarker and/ordetecting the mutation in the biomarker is known to those of ordinaryskill, for example by performing the steps outlined in the commerciallyavailable assay kit sold by Sigma-Aldrich, Thermo Fisher Scientific, R &D Systems, ZeptoMetrix Inc., Cayman Inc., Abcam, Trevigen, DojindoMolecular Technologies, Biovision, and Enzo Life Sciences.

In some embodiments, a concentration of the biomarker is measured beforeand after the administration. When the concentration of the biomarker ismaintained, the method may further comprise increasing the effectiveamount of the compound of the present disclosure by at least 5%, atleast 10%, or at least 30%, up to 50%, up to 60%, or up to 80% of aninitial effective amount that is in a range of 0.1-500 mg/kg per bodyweight of the subject. The increased effective amount may be in a rangeof 0.105-900 mg/kg, preferably 1-500 mg/kg, more preferably 10-250mg/kg. The subject may be administered with the increased dosage for alonger period (e.g. 1 week more, 2 weeks more, or 2 months more) thanthe duration prescribed with the initial effective amount.

In some embodiments, the mutation in the biomarker is detected beforeadministering the composition to identify subjects predisposed to thedisease. Alternatively, the biomarkers are measured/detected after eachadministration. For example, the measurement may be 1-5 minutes, 1-30minutes, 30-60 minutes, 1-2 hours, 2-12 hours, 12-24 hours, 1-2 days,1-15 weeks, 15-20 weeks, 20-30 weeks, 30-40 weeks, 40-50 weeks, 1 year,2 years, or any period of time in between after the administration.

In some embodiments, the administration is stopped once the subject istreated.

The examples below are intended to further illustrate protocols forpreparing, characterizing the compounds of formulae (I), (II), and(III), and uses thereof, and are not intended to limit the scope of theclaims.

Example 1 Chemical Synthesis: Overview

First, 2-(4-nitrophenyl)acetic acid 1 was stirred with oxalyl chloridein dichloromethane (DCM) and a catalytic amount of N,N-dimethylformamide(DMF) at room temperature for 4 h to afford 2-(4-nitrophenyl) acetylchloride 2. In an ice bath, intermediates 3a-j were prepared by thereaction of 2-(4-nitrophenyl) acetyl chloride 2 and the correspondingamine in the presence of diisopropylethylamine (DIPEA) in DCM. Thepivotal amines 4a-j were obtained by reduction of the nitro followingone of two techniques. One method was heating the nitro intermediatewith SnCl₂ dihydrate in ethyl acetate for 4 h. Alternatively, the amineswere prepared using flow chemistry reduction instrument H-Cube Pro™(Thales Technology, Hungary) with 10% Pd—C cartridge to catalyze thereduction in high pressure (10 atm.) and temperature (40° C.) (see FIG.4).

To prepare the oxazolone intermediates (7a-i) for the second portion ofKIM compounds, we employed Erlenmeyer chemistry by reacting eitherN-acetylglycine (5a) or hippuric acid (5b) with the appropriatealdehydes 6a-e. The final step involved reaction of the amine (4a-j)with the corresponding oxazolone (7a-i) in dry pyridine to produce KIM-C(imidazolones) compounds. Alternatively, the oxazolones (7a-i) wereheated with the appropriate amine (4a-j) in acetonitrile to give KIM-Vcompounds (see FIG. 5). The truncated analogue KIM-112V was prepared byreacting oxazolone 7b with p-toluidine under the same conditionsmentioned above for the preparation of KIM-V series.

All compounds were characterized by NMR spectroscopy and their molecularformulae were established using HRMS. The purities of the compounds weredetermined to be higher than 95% using LC/MS.

Example 2 Chemical Synthesis: Experimental

All melting points were uncorrected and measured using the capillarymelting point instrument BI 9100 (Barnstead Electrothermal, UK).Infrared spectra were recorded on a Thermo Scientific Niccolet iS10FT-IR Spectrometer (King Fahd Center for Medical Research, KingAbdulaziz University, Jeddah, Saudi Arabia). In this disclosure, onlycharacteristic IR stretching bands were listed, including NH, OH, CH,C═O, C═N, and/or C═C. In FT-IR, all samples were measured neat. ¹H NMRspectra were recorded on an AVANCE-III 600 MHz and AVANCE-III HD 850 MHzspectrometers (Bruker, Germany), and chemical shifts were expressed asppm against TMS as an internal reference (King Fand Center for MedicalResearch and Faculty of Science, King Abdulaziz University, Jeddah,Saudi Arabia). LC/MS analysis was performed on an Agilent 6320 Ion TrapHPLC-ESI-MS/DAD (Santa Clara, Calif., USA) with the following settings.The analytes were separated using a Macherey-Nagel Nucleodur-C18 column(150 mm length×4.6 mm i.d., 5 μm) (Macherey-Nagel GMBH & Co. KG, Duren,Germany). Mobile phase, isocratic elution using a mixture ofacetonitrile and 0.01 formic acid in water (80: 20, v/v). The flow ratewas 0.4 mL/min; total run time=20 min. Purities were reported accordingto percentage of Peak Areas at a wavelength of 280 nm. High-resolutionmass spectrometry (HRMS) was performed in the Faculty of Science, KingAbdulaziz University on Impact II™ Q-TOF spectrometer (Bruker, Germany).Column chromatography was performed on a silica gel 60 (particle size0.06 mm-0.20 mm).

Example 3 General Procedure for Preparation of Amines 4a-4j

A mixture of 2-(4-nitrophenyl)acetic acid (10 mmol, 1.81 g) and 50 mLdichloromethane (DCM) was placed in a dry 3-neck round bottom flask,flushed with nitrogen, and stirred in an ice bath. Then, oxalyl chloride(11.6 mmol, 1.48 g, 1 mL) in 5 mL DCM was placed in an addition funneland was then fast-dropped to the original mixture. After the addition ofoxalyl chloride was completed, 1 drop of dimethylformamide (DMF) wasadded. 15 min later, the ice bath was removed and the mixture wasstirred at room temperature (r.t.) for 4 hours as all the acid dissolvedcompletely. The solvent was removed using rotary evaporator and theresidue 2-(4-nitrophenyl)acetyl chloride 2 was collected, dissolved in40 mL DCM, and stirred in an ice bath for 10 min. The appropriate amine(a-j, FIG. 4) (10 mmol, 1.1 g, 1.1 mL) was added using an additionfunnel, along with diisopropylethylamine (DIPEA) (10 mmol, 1.55 g, 2.1mL) in 40 mL DCM, which was added drop wise to the acid chloride. Afterthe ice melted, the stirring continued overnight at room temperature.The solid particles were collected by filtration, and washed with asmall amount of DCM. Yellowish white crystalline solid was observed. Thecompletion of the reaction was checked by TLC for both the solid andfiltrate using ethyl acetate hexane mixture in the ratio of 1:1 againstthe starting materials. The filtrate was neutralized by HCl (1 M) andthe organic layer was collected, dried using sodium sulfate. After thesolvent was rotavaped, a solid (3a-j) was collected and washed withether.

In a 150 mL round bottom flask rapped with Aluminum foil, a mixture ofN-benzyl-2-(4-nitrophenyl)acetamide (6.58 mmol, 1.78 g), SnCl₂ dihydrate(26.34 mmol, 5.95 g), ethyl acetate (40 mL), and water (0.5 mL) wasrefluxed for 4 hours. The mixture was then cooled, diluted by ethylacetate (40 mL), and treated with a cold solution of 40% NaOH (80 mL),thereby forming an emulsion. Fresh water was added to break theemulsion. The organic layer was separated. The aqueous layer was washedwith 10 mL of ethyl acetate, then combined with the organic layer. Thecombined layers were washed with 15 mL of brine and dried with magnesiumsulfate. The ethyl acetate was removed using rotary evaporator and asolid product was collected.

Example 4 Characterization of Amines 4a-4j2-(4-aminophenyl)-N-benzylacetamide (4a)

The amine 4a was synthesized according to the procedure above and itscharacterization data were found similar to literature [Kumar, M.;Sharma, S.; Thakur, K.; Nayal, O. S.; Bhatt, V.; Thakur, M. S.; Kumar,N.; Singh, B.; Sharma, U., Montmorillonite-K10-CatalyzedMicrowave-Assisted Direct Amidation of Unactivated Carboxylic Acids withAmines: Maintaining Chiral Integrity of Substrates. Asian J. Org. Chem.2017, 6 (3), 342-346, incorporated herein by reference in its entirety].Melting point: 140-142° C. Yield: 1.55 g (98.7%). The compound was usedfor the next step without further characterization.

2-(4-aminophenyl)-N-phenylacetamide (4b)

¹H NMR (600 MHz, CDCl₃) δ 7.41 (d, J=7.53 Hz, 2H), 7.21-7.35 (m, 2H),7.04-7.20 (m, 2H), 6.73 (d, J=7.91 Hz, 1H), 3.74 (br. s., 1H), 3.64 (s,1H).

2-(4-aminophenyl)-N-(4-methoxybenzyl)acetamide (4c)

¹H NMR (600 MHz, CDCl₃) δ 7.10 (d, J=8.66 Hz, 1H), 7.02 (d, J=8.28 Hz,1H), 6.78-6.92 (m, 1H), 6.61-6.75 (m, 1H), 5.71 (br. s., 1H), 4.32 (d,J=5.65 Hz, 1H), 3.75-3.86 (m, 2H), 3.67 (br. s., 1H), 3.44-3.59 (m, 1H).

2-(4-aminophenyl)-N-(furan-2-ylmethyl)acetamide (4d)

¹H NMR (600 MHz, CDCl₃) δ 7.02 (d, J=8.28 Hz, 2H), 6.65 (d, J=8.28 Hz,2H), 6.28 (br. s., 1H), 6.13 (br. s., 1H), 5.78 (br. s., 1H), 4.38 (d,J=5.65 Hz, 2H), 3.70 (br. s., 2H), 3.43-3.60 (m, 2H).

2-(4-aminophenyl)-N-phenethylacetamide (4f)

¹H NMR (600 MHz, CDCl₃) δ 7.17-7.34 (m, 3H), 7.05 (d, J=7.53 Hz, 2H),6.94 (d, J=8.28 Hz, 2H), 6.63 (d, J=7.91 Hz, 2H), 5.43 (br. s., 1H),3.70 (br. s., 1H), 3.38-3.56 (m, 4H), 2.68-2.81 (m, 2H). ¹³C NMR (151MHz, CDCl₃) δ 171.7, 145.6, 138.8, 130.5, 128.7, 128.5, 126.4, 124.4,115.6, 43.0, 40.7, 35.6.

2-(4-aminophenyl)-N-(1-phenylethyl)acetamide (4 g)

¹H NMR (600 MHz, CDCl₃) δ 7.28 (d, J=11.67 Hz, 1H), 7.17-7.26 (m, 2H),7.03 (d, J=7.91 Hz, 2H), 6.67 (d, J=8.28 Hz, 2H), 5.65 (br. s., 1H),5.13 (dd, J=6.96, 14.12 Hz, 1H), 3.70 (br. s., 1H), 3.46-3.55 (m, 2H),1.35-1.46 (m, 3H).

2-(4-aminophenyl)-N-(4-fluorobenzyl)acetamide (4h)

¹H NMR (600 MHz CDCl₃) δ 7.10-7.22 (m, 2H), 7.03 (d, J=8.28 Hz, 2H),6.93-7.01 (m, 1H), 6.66 (d, J=8.28 Hz, 2H), 5.74 (br. s., 1H), 4.36 (d,J=5.65 Hz, 2H), 3.69 (br. s., 2H), 3.52 (s, 2H).

N-([1,1′-biphenyl]-4-ylmethyl)-2-(4-aminophenyl)acetamide (4i)

¹H NMR (600 MHz, CDCl₃) δ 7.50-7.64 (m, 3H), 7.41-7.50 (m, 2H), 7.35 (d,J=7.15 Hz, 1H), 7.22-7.32 (m, 4H), 7.06 (d, J=7.91 Hz, 1H), 6.67 (d,J=8.28 Hz, 1H), 5.76 (br. s., 1H), 4.45 (d, J=6.02 Hz, 1H), 3.68 (br.s., 1H), 3.55 (s, 1H).

2-(4-aminophenyl)-N-(3-fluorobenzyl)acetamide (4j)

¹H NMR (600 MHz, CDCl₃) δ 7.21-7.34 (m, 1H), 7.05 (d, J=7.91 Hz, 2H),6.90-7.02 (m, 2H), 6.88 (d, J=9.79 Hz, 1H), 6.67 (d, J=8.28 Hz, 2H),5.77 (br. s., 1H), 4.39 (d, J=6.02 Hz, 2H), 3.70 (br. s., 1H), 3.52-3.61(m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 171.8, 163.8, 145.8, 141.0, 130.5,130.1, 130.1, 124.2, 122.9, 115.7, 114.3, 114.1, 43.0, 42.9.

Example 5 General Procedure for the Preparation of(Z)-4-Arylidene-2-Substituted Oxazol-5(4H)-One Intermediates (7a-i)

The acylglycine 5a or 5b (10 mmol) was mixed with an equimolar amount ofappropriate aldehyde 6a-e, acetic anhydride (1.9 mL, 2 equiv.) andfreshly dried sodium acetate (0.08 g, 0.1 equiv.). The mixture washeated at 80° C. for 30 min then cooled. The resulting solid was washedwith water and sodium bicarbonate solution, and dried by vacuumfiltration. The yellow solid was washed with petroleum ether severaltimes and used without further purification for the next step.

Example 6 General Procedure for the Synthesis of KIM-C Derivatives

The appropriate oxazolone 7a-i (2 mmol) was mixed with an amine 4a (0.48g, 2 mmol) in dry pyridine (6 mL) under inert atmosphere. The mixturewas heated in Integrity Stem 10 Reactor (Cole Parmer, England) at 100°C. for 8 hours. The mixture was cooled and poured into ice/HCl mixture.The precipitated solid was collected by filtration, washed with water,and purified by silica gel chromatography (gradient petroleum-ether to10% ethyl acetate in petroleum ether).

Example 7(Z)—N-benzyl-2-(4-(4-benzylidene-2-methyl-5-oxo-4,5-dihydro-JH-imidazol-1-yl)phenyl)acetamide(KIM-111C)

This compound was off-white solid, m.p. 164-165° C.; ¹H NMR (600 MHz,CDCl₃) δ 8.19 (d, J=6.78 Hz, 2H), 7.41-7.52 (m, 5H), 7.34 (t, J=7.15 Hz,2H), 7.20-7.32 (m, 6H), 6.03 (br. s., 1H), 4.46 (d, J=5.65 Hz, 2H), 3.65(br. s., 2H).

Example 8(Z)—N-benzyl-2-(4-(4-(4-chlorobenzylidene)-2-methyl-5-oxo-4,5-dihydro-JH-imidazol-1-yl)phenyl)acetamide(KIM-121C)

This compound was off-white solid, m.p. 209-211° C.; ¹H NMR (600 MHz,CDCl₃) δ 8.12-8.18 (m, 2H), 7.45 (d, J=8.28 Hz, 2H), 7.47 (d, J=7.91 Hz,2H), 7.32-7.37 (m, 2H), 7.23-7.32 (m, 6H), 7.17 (s, 1H), 5.88 (br. s.,1H), 4.44-4.50 (m, 2H), 3.65-3.71 (m, 2H), 2.35 (s, 3H).

Example 9(Z)—N-benzyl-2-(4-(4-(4-hydroxy-3-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-131C)

This compound was off-white solid, m.p. 199-200° C.; ¹H NMR (600 MHz,CDCl₃) δ 8.05 (s., 1H), 7.57 (d, J=7.53 Hz, 1H), 7.44 (d, J=7.53 Hz,2H), 7.33 (d, J=7.15 Hz, 2H), 7.20-7.31 (m, 5H), 7.15 (s, 1H), 6.98 (d,J=8.28 Hz, 1H), 6.03 (br. s., 1H), 5.11 (br. s., 1H), 4.44-4.49 (d, 2H),4.01 (s., 3H), 3.67 (s, 2H), 3.49 (br. s., 2H), 2.30 (s, 3H).

Example 10(Z)—N-benzyl-2-(4-(4-(4-methoxybenzylidene)-2-methyl-5-oxo-4,5-dihydro-JH-imidazol-1-yl)phenyl)acetamide(KIM-161C)

This compound was off-white solid, m.p. 185° C.; ¹H NMR (600 MHz, CDCl₃)δ 8.18 (d, J=8.66 Hz, 2H), 7.44 (d, J=7.91 Hz, 2H), 7.32-7.37 (m, 2H),7.22-7.32 (m, 5H), 7.18 (s, 1H), 7.00 (d, J=8.66 Hz, 2H), 5.97 (br. s.,1H), 4.46 (d, J=5.65 Hz, 2H), 3.89 (s, 3H), 3.66 (s, 2H), 2.30 (s, 3H).

Example 11(Z)—N-benzyl-2-(4-(4-benzylidene-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-211C)

This compound was yellow solid, m.p. 218-219° C.; ¹H NMR (600 MHz,CDCl₃) δ 8.24-8.28 (m, 2H), 7.57 (dd, J=1.13, 8.28 Hz, 2H), 7.44-7.47(m, 3H), 7.37 (d, J=8.28 Hz, 2H), 7.28-7.35 (m, 7H), 7.23-7.26 (m, 2H),7.16-7.19 (m, 2H), 5.81 (br. s., 1H), 4.45-4.48 (m, 2H), 3.67 (s, 2H).

Example 12(Z)—N-benzyl-2-(4-(4-(4-chlorobenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-221C)

This compound was pale yellow solid, m.p. 218-220° C.; ¹H NMR (600 MHz,CDCl₃) δ 8.24-8.28 (m, 2H), 7.57 (dd, J=1.13, 8.28 Hz, 2H), 7.45-7.47(m, 2H), 7.37 (d, J=8.28 Hz, 2H), 7.29-7.35 (m, 6H), 7.23-7.26 (m, 2H),7.16-7.19 (m, 2H), 5.81 (br. s., 1H), 4.47 (d, J=5.65 Hz, 2H), 3.67 (s,2H).

Example 13(Z)—N-benzyl-2-(4-(4-(4-hydroxy-3-methoxybenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-231C)

This compound was pale orange-yellow solid, m.p. 109-112° C.; ¹H NMR(600 MHz, CDCl₃) δ 8.28 (s, 1H), 7.73 (d, J=8.28 Hz, 1H), 7.55 (d,J=7.91 Hz, 2H), 7.44 (dd, J=7.40 and 7.24 Hz, 1H), 7.32-7.39 (m, 4H),7.26-7.31 (m, 4H), 7.24 (d, J=7.15 Hz, 2H), 7.18 (d, J=6.40 Hz, 2H),7.14 (d, J=7.91 Hz, 1H), 5.85 (br. s., 1H), 4.43-4.48 (m, 2H), 4.03 (br.s., 1H), 3.96 (s, 3H), 3.67 (s, 2H).

Example 14(Z)—N-benzyl-2-(4-(4-(furan-2-ylmethylene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-241C)

This compound was pale orange-yellow solid, m.p. 199-200° C.; ¹H NMR(600 MHz, CDCl₃) δ 7.68-7.72 (m, 1H), 7.53-7.61 (m, 3H), 7.40-7.49 (m,1H), 7.31-7.39 (m, 4H), 7.21-7.31 (m, 6H), 7.14-7.20 (m, 2H), 6.66 (dd,J=2.07, 3.20 Hz, 1H), 5.85 (br. s., 1H), 4.43-4.49 (m, 2H), 3.66 (s,2H).

Example 15(Z)—N-benzyl-2-(4-(4-(4-methoxybenzylidene)-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl)phenyl)acetamide(KIM-261C)

This compound was pale orange-yellow solid, m.p. 193° C.; ¹H NMR (600MHz, CDCl₃) δ 8.30 (d, J=9.03 Hz, 2H), 7.52-7.58 (m, 2H), 7.39-7.45 (m,1H), 7.26-7.37 (m, 8H), 7.24 (d, J=7.53 Hz, 2H), 7.14-7.20 (m, 2H), 7.02(d, J=9.03 Hz, 2H), 5.84 (br. s., 1H), 4.43-4.48 (m, 2H), 3.88-3.93 (m,3H), 3.67 (br. s., 2H).

Example 16 General Procedure for Synthesis of KIM-V Compounds

The appropriate oxazolone 7a-i (2 mmol) was mixed with the amine 4a-j(0.48 g, 2 mmol) in acetonitrile (6 mL) under inert atmosphere. Themixture was heated to 80° C. for 8 hours in Integrity Stem 10™ Reactor(Cole Parmer, England). The mixture was cooled and poured into anice/HCl mixture. The precipitated solid was collected by filtration,washed with water, and purified by silica gel chromatography (gradientdichloromethane to 5% MeOH in dichloromethane).

Example 17(E)-2-acetamido-N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-3-phenylacrylamide(KIM-111V)

The purified product KIM-111V was a white solid, m.p. 197° C. IR (KBr,ν_(max) cm⁻¹) 3236, 3086, 1659, 1638, 1599, 1533, 1505, 1270; ¹H NMR ¹HNMR (600 MHz, DMSO-d₆) δ 7.61 (d, J=7.91 Hz, 1H), 7.64 (d, J=8.66 Hz,2H), 7.40-7.48 (m, 1H), 7.29-7.39 (m, 2H), 7.21-7.28 (m, 3H), 4.25-4.32(m, 2H), 3.45 (br. s., 1H), 2.04 (s, 2H); LC-MS (ESI), m/z 428.1969[M+H]⁺. Purity: 97%.

Example 18(E)-2-acetamido-N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-3-(4-chlorophenyl)acrylamide(KIM-121V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7b (2 mmol, 0.443 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.529 g). The productKIM-121V was white solid, m.p.>300° C. (dec); IR (KBr, ν_(max) cm⁻¹)3243, 3065, 1724, 1647, 1546, 1395, 1329; ¹H NMR (600 MHz, DMSO-d₆) δ7.60-7.71 (m, 2H), 7.40-7.51 (m, 2H), 7.29-7.34 (m, 2H), 7.18-7.29 (m,3H), 4.28 (d, J=5.65 Hz, 1H), 3.45 (s, 1H), 2.02 (s, 1H), 1.63 (d,J=1.88 Hz, 2H); ¹³C NMR (151 MHz, DMSO-d₆) δ 175.1, 134.2, 131.8, 131.5,130.7, 129.7, 129.6, 129.2, 129.0, 128.1, 127.9, 127.7, 127.4, 120.6,40.6, 40.5, 40.3, 40.2, 40.0, 39.9, 39.7, 25.4; Purity 93%.

Example 19(E)-2-acetamido-N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide (KIM-131V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7c (2 mmol, 0.446 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.529 g; ¹H NMR (600 MHz,Acetone) δ 9.38 (s, 1H), 8.97 (s, 1H), 7.70 (d, J=8.66 Hz, 1H),7.33-7.35 (m, 1H), 7.26-7.32 (m, 5H), 7.23 (d, J=6.78 Hz, 1H), 7.15 (d,J=1.88 Hz, 1H), 7.06-7.10 (m, 2H), 4.40 (d, J=6.02 Hz, 2H), 3.86 (s,2H), 3.55 (s, 2H), 2.28 (s, 3H), 2.16 (s, 2H), 1.31 (br. s., 1H).

Example 20(E)-2-acetamido-N-(4-(2-(benzylamino)-2-oxoethyl)phenyl)-3-(4-methoxyphenyl)acrylamide (KIM-161V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7d (2 mmol, 0.434 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.529 g). The productKIM-161V was white solid, m.p. 189-200° C.; ¹H NMR (600 MHz, DMSO-d₆) δ7.63 (d, J=8.66 Hz, 2H), 7.58 (d, J=8.66 Hz, 2H), 7.29-7.36 (m, 3H),7.21-7.27 (m, 6H), 6.97-7.03 (m, 3H), 4.28 (d, J=6.02 Hz, 2H), 3.80 (s,4H), 3.45 (s, 2H), 2.05 (s, 3H); ¹³C NMR (151 MHz, DMSO) δ 171.0, 168.9,140.1, 138.4, 131.6, 129.9, 129.0, 127.9, 127.5, 119.6, 42.9, 42.5,40.7, 40.6, 40.5, 40.3, 40.2, 40.0, 39.9, 39.8, 24.6.

Example 21(E)-N-(3-((4-(2-(benzylamino)-2-oxoethyl)phenyl)amino)-3-oxo-1-phenylprop-1-en-2-yl)benzamide(KIM-211V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7e (2 mmol, 0.498 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.498 g). The productKIM-211V was white solid, m.p. 214-216° C.; ¹H NMR (600 MHz, DMSO-d₆) δ10.11 (s, 1H), 10.14 (s, 1H), 8.51 (br. s., 1H), 8.00-8.06 (m, J=7.53Hz, 2H), 7.62-7.69 (m, 4H), 7.61 (d, J=6.78 Hz, 1H), 7.54 (t, J=7.34 Hz,2H), 7.37-7.43 (m, 2H), 7.28-7.36 (m, 3H), 7.21-7.27 (m, 5H), 7.17 (s,1H), 4.28 (d, J=5.65 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃) δ 175.8, 171.5,169.7, 169.7, 144.9, 143.1, 139.7, 138.9, 136.8, 136.4, 135.0, 134.5,134.4, 134.1, 133.9, 133.8, 133.4, 133.3, 132.6, 132.2, 132.2, 125.6,125.4, 47.6, 47.3.

Example 22(E)-N-(3-((4-(2-(benzylamino)-2-oxoethyl)phenyl)amino)-1-(4-chlorophenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-221V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.498 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.567 g). The productKIM-221V was white solid, m.p. 255° C.; IR (KBr, ν_(max) cm⁻¹) 3283,1684, 1638, 1535, 1480, 1311; ¹H NMR (600 MHz, DMSO-d₆) δ 10.20 (br. s.,1H), 10.15 (br. s., 1H), 8.51 (d, J=5.65 Hz, 1H), 8.03 (d, J=7.15 Hz,2H), 7.59-7.70 (m, 5H), 7.52-7.58 (m, 2H), 7.45-7.50 (m, 2H), 7.32 (t,J=7.53 Hz, 2H), 7.24 (d, J=6.78 Hz, 4H), 7.13 (br. s., 1H), 4.28 (d,J=5.27 Hz, 2H), 3.46 (br. s., 2H).

Example 23(E)-N-(3-((4-(2-(benzylamino)-2-oxoethyl)phenyl)amino)-1-(4-hydroxy-3-methoxyphenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-231V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7 g (2 mmol, 0.498 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.590 g). The productKIM-231V was white solid, m.p. 186-188° C.; ¹H NMR (600 MHz, DMSO-d₆) δ9.89 (br. s., 1H), 8.50 (br. s., 1H), 7.50 (d, J=8.28 Hz, 3H), 7.28-7.35(m, 3H), 7.17-7.27 (m, 6H), 4.27 (d, J=5.65 Hz, 3H), 3.42 (s, 3H), 2.04(s, 4H); ¹³C NMR (151 MHz, Acetone) δ 168.2, 167.0, 164.9, 163.9, 161.3,151.8, 151.5, 142.7, 139.1, 133.8, 132.7, 130.4, 129.5, 128.4, 127.8,126.0, 123.6, 115.8, 115.2, 55.7, 19.9.

Example 24(E)-N-(3-((4-(2-(benzylamino)-2-oxoethyl)phenyl)amino)-1-(4-methoxyphenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-261V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7i (2 mmol, 0.558 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.590 g). The productKIM-261V was white solid, mp 186-188° C.; ¹H NMR (600 MHz, DMSO-d₆) δ9.88 (br. s., 1H), 8.50 (d, J=5.27 Hz, 1H), 7.50 (d, J=8.28 Hz, 3H),7.31 (t, J=7.53 Hz, 3H), 7.22-7.27 (m, 4H), 7.19 (d, J=8.28 Hz, 3H),4.27 (d, J=6.02 Hz, 3H), 3.42 (s, 3H), 2.04 (s, 4H); ¹³C NMR (151 MHz,DMSO) δ 171.0, 168.9, 140.1, 138.4, 131.6, 129.9, 129.0, 127.9, 127.5,119.6, 42.9, 42.5, 24.6.

Example 25(E)-N-(3-((4-(2-(benzylamino)-2-oxoethyl)phenyl)amino)-1-(4-fluoro-2-methylphenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-2101V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7j (2 mmol, 0.562 g) and2-(4-aminophenyl)-N-benzylacetamide (2.2 mmol, 0.590 g). The productKIM-2101V was white solid, m.p. 156° C.; ¹H NMR (600 MHz, Acetone) δ8.07 (d, J=7.53 Hz, 2H), 7.72 (dd, J=2.64, 8.66 Hz, 2H), 7.67 (d, J=9.79Hz, 2H), 7.59-7.65 (m, 3H), 7.53 (t, J=7.72 Hz, 2H), 7.41 (d, J=8.66 Hz,2H), 7.33 (d, J=8.66 Hz, 2H), 7.30 (br. s., 1H), 7.26-7.29 (m, 2H), 7.05(t, J=7.91 Hz, 1H), 3.67 (s, 2H).

Example 26(E)-N-(1-(4-chlorophenyl)-3-oxo-3-((4-(2-oxo-2-(phenylamino)ethyl)phenyl)amino)prop-1-en-2-yl)benzamide(KIM-22101V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.567 g) and2-(4-aminophenyl)-N-phenylacetamide (2.2 mmol, 0.497 g). The productKIM-22101V was white solid, m.p. 166-175° C.; ¹H NMR (600 MHz, Acetone)δ 8.07 (d, J=7.53 Hz, 2H), 7.72 (dd, J=2.64, 8.66 Hz, 2H), 7.67 (d,J=9.79 Hz, 2H), 7.59-7.65 (m, 3H), 7.53 (t, J=7.72 Hz, 2H), 7.41 (d,J=8.66 Hz, 2H), 7.33 (d, J=8.66 Hz, 2H), 7.30 (br. s., 1H), 7.26-7.29(m, 2H), 7.05 (t, J=7.91 Hz, 1H), 3.67 (s, 2H); ¹³C NMR (151 MHz, DMSO)δ 169.7, 166.7, 164.3, 140.1, 138.5, 134.4, 134.2, 132.6, 132.1, 131.9,131.6, 130.7, 130.1, 130.0, 129.3, 129.1, 128.5, 128.3, 128.0, 123.9,120.7, 119.8, 43.9.

Example 27(E)-N-(1-(4-chlorophenyl)-3-((4-(2-((4-methoxybenzyl)amino)-2-oxoethyl)phenyl)amino)-3-oxoprop-1-en-2-yl)benzamide(KIM-2216V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.567 g) and2-(4-aminophenyl)-N-(4-methoxybenzyl)acetamide (2.2 mmol, 0.594 g). Theproduct KIM-2216V was white solid, m.p. 166-175° C.; ¹H NMR (600 MHz,Acetone) δ 7.69 (d, J=8.66 Hz, 2H), 7.58-7.66 (m, 2H), 7.54 (t, J=7.53Hz, 2H), 7.42 (d, J=8.28 Hz, 3H), 7.24-7.34 (m, 3H), 7.19 (d, J=8.66 Hz,2H), 6.84-6.88 (m, 2H), 4.31 (s, 2H), 3.77 (s, 3H), 2.82 (br. s., 5H);NMR (151 MHz, DMSO) δ 170.8, 166.7, 164.2, 159.5, 138.4, 134.4, 134.2,132.6, 132.3, 131.6, 130.7, 130.0, 129.3, 129.1, 128.5, 127.9, 120.6,120.5, 114.3, 55.3, 43.0, 42.7.

Example 28(E)-N-(1-(4-chlorophenyl)-3-((4-(2-((furan-2-ylmethyl)amino)-2-oxoethyl)phenyl)amino)-3-oxoprop-1-en-2-yl)benzamide(KIM-2230V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.567 g) and2-(4-aminophenyl)-N-(furan-2-ylmethyl)acetamide (2.2 mmol, 0.506 g). Theproduct KIM-2230V was white solid, m.p. 217-218° C.; ¹H NMR (600 MHz,Acetone) δ 7.67-7.84 (m, 2H), 7.58-7.66 (m, 2H), 7.54 (t, J=7.72 Hz,3H), 7.38-7.48 (m, 3H), 7.22-7.37 (m, 3H), 4.34-4.49 (m, 2H), 3.48-3.66(m, 2H), 2.84 (br. s., 4H); ¹³C NMR (151 MHz, CDCl₃) δ 156.1, 155.2,154.9, 143.1, 142.8, 142.5, 142.5, 140.9, 140.2, 139.8, 139.0, 138.7,137.2, 132.2, 130.7, 130.2, 129.4, 129.2, 129.2, 129.1, 127.8, 121.0,110.7, 110.2, 107.7, 43.2, 36.9.

Example 29(E)-N-(3-((4-(2-(benzyl(methyl)amino)-2-oxoethyl)phenyl)amino)-1-(4-chlorophenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-2245V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.567 g) and2-(4-aminophenyl)-N-benzyl-N-methylacetamide (2.2 mmol, 0.559 g). Theproduct KIM-2245 was white solid, m.p. 112° C.; ¹H NMR (600 MHz,Acetone) δ 8.07 (d, J=7.53 Hz, 2H), 7.66-7.86 (m, 2H), 7.57-7.66 (m,3H), 7.47-7.57 (m, 2H), 7.35-7.47 (m, 3H), 7.29-7.35 (m, 2H), 7.19-7.29(m, 5H), 4.66 (s, 1H), 4.60 (s, 1H), 3.79 (s, 1H), 3.75 (s, 1H), 2.99(s, 2H), 2.88 (s, 1H); ¹³C NMR (151 MHz, DMSO) δ 171.5, 171.4, 166.8,164.5, 138.8, 138.4, 138.2, 134.4, 134.2, 132.6, 132.3, 131.7, 131.6,129.8, 129.4, 129.3, 129.1, 129.1, 128.5, 128.5, 128.0, 127.9, 127.7,127.4, 120.8, 120.7, 53.8, 50.9, 40.5, 40.2, 35.3, 33.7.

Example 30(E)-N-(1-(4-chlorophenyl)-3-oxo-3-((4-(2-oxo-2-(phenethylamino)ethyl)phenyl)amino)prop-1-en-2-yl)benzamide(KIM-2246V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.567 g) and2-(4-aminophenyl)-N-phenethylacetamide (2.2 mmol, 0.540 g). The productKIM-2246V was white solid, m.p. 135° C.; ¹H NMR (600 MHz, Acetone) δ9.58 (s, 1H), 8.08 (d, J=7.91 Hz, 2H), 7.68 (dd, J=2.45, 8.47 Hz, 2H),7.60-7.65 (m, 3H), 7.51-7.56 (m, 2H), 7.39-7.43 (m, 2H), 7.24-7.30 (m,3H), 7.15-7.24 (m, 5H), 3.39-3.45 (m, 4H), 2.77 (t, J=7.34 Hz, 2H).

Example 31(E)-N-(1-(4-chlorophenyl)-3-oxo-3-((4-(2-oxo-2-((1-phenylethyl)amino)ethyl)phenyl)amino)prop-1-en-2-yl)benzamide (KIM-2247V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.539 g) and2-(4-aminophenyl)-N-(1-phenylethyl)acetamide (2.2 mmol, 0.540 g). Theproduct KIM-2247V was white solid, m.p. 192° C.; ¹H NMR (600 MHz,Acetone) δ 8.07 (d, J=7.53 Hz, 2H), 7.68 (d, J=8.28 Hz, 2H), 7.59-7.65(m, 3H), 7.53 (t, J=7.72 Hz, 2H), 7.41 (d, J=8.66 Hz, 2H), 7.18-7.37 (m,8H), 5.07 (q, J=7.15 Hz, 1H), 3.50 (s, 2H), 1.42 (d, J=6.78 Hz, 3H); ¹³CNMR (151 MHz, DMSO) δ 170.0, 145.3, 134.4, 134.3, 134.2, 132.6, 132.4,132.2, 131.6, 130.7, 129.9, 129.3, 129.1, 129.0, 128.9, 128.5, 128.3,128.0, 127.3, 126.7, 120.6, 120.4, 49.0, 43.0, 22.4.

Example 32(E)-N-(1-(4-chlorophenyl)-3-((4-(2-((4-fluorobenzyl)amino)-2-oxoethyl)phenyl)amino)-3-oxoprop-1-en-2-yl)benzamide(KIM-2248V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.539 g) and2-(4-aminophenyl)-N-(4-fluorobenzyl)acetamide (2.2 mmol, 0.568 g). Theproduct KIM-2248V was white solid, m.p. 216° C.; ¹H NMR (600 MHz,Acetone) δ 8.08 (d, J=7.53 Hz, 2H), 7.68-7.72 (m, 2H), 7.63 (d, J=8.28Hz, 3H), 7.54 (t, J=7.72 Hz, 2H), 7.41 (d, J=8.28 Hz, 2H), 7.31 (dd,J=5.65, 8.66 Hz, 3H), 7.26-7.29 (m, 3H), 7.05 (t, J=8.85 Hz, 2H), 4.37(s, 2H), 3.54 (s, 2H).

Example 33(E)-N-(3-((4-(2-(([1,1′-biphenyl]-4-ylmethyl)amino)-2-oxoethyl)phenyl)amino)-1-(4-chlorophenyl)-3-oxoprop-1-en-2-yl)benzamide(KIM-2249V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.539 g) andN-([1,1′-biphenyl]-4-ylmethyl)-2-(4-aminophenyl)acetamide (2.2 mmol,0.695 g). The product KIM-2249V was white solid, m.p. 242° C.; ¹H NMR(600 MHz, Acetone) δ 9.53 (s, 1H), 8.07 (d, J=7.91 Hz, 1H), 7.71 (d,J=8.66 Hz, 1H), 7.66 (s, 1H), 7.64 (d, J=2.64 Hz, 1H), 7.63 (s, 1H),7.60 (s, 1H), 7.58 (s, 1H), 7.52-7.56 (m, 1H), 7.46 (t, J=7.91 Hz, 2H),7.42 (d, J=8.66 Hz, 1H), 7.37 (d, J=7.91 Hz, 2H), 7.31 (d, J=8.28 Hz,1H), 7.28 (s, 1H), 4.45 (d, J=6.02 Hz, 1H), 3.57 (s, 1H), 2.84 (s, 5H),2.81 (s, 3H).

Example 34(E)-N-(1-(4-chlorophenyl)-3-((4-(2-((3-fluorobenzyl)amino)-2-oxoethyl)phenyl)amino)-3-oxoprop-1-en-2-yl)benzamide(KIM-2250V)

This compound was prepared according to the procedure described for thesynthesis of KIM-111V starting from oxazolone 7f (2 mmol, 0.539 g) and2-(4-aminophenyl)-N-(3-fluorobenzyl)acetamide (2.2 mmol, 0.567 g). Theproduct KIM-2250V was white solid, mp 242° C.; ¹H NMR (600 MHz, Acetone)δ 8.08 (d, J=7.91 Hz, 2H), 7.71 (d, J=8.66 Hz, 2H), 7.60-7.66 (m, 3H),7.52-7.58 (m, 2H), 7.39-7.44 (m, J=8.28 Hz, 2H), 7.27-7.36 (m, 4H), 7.11(d, J=7.15 Hz, 1H), 7.03-7.07 (m, 1H), 6.99 (dt, J=2.45, 8.56 Hz, 1H),4.41 (s, 2H), 3.56 (s, 2H).

Example 35 Methods for Cytotoxic Activities Against MCF7 and PC3: CellCulture

MCF-7 (breast) and PC3 (prostate) cancer cells were each grown inRPMI-1640 medium supplemented with 10% heat inactivated FBS, 50 units/mLof penicillin, and 50 mg/mL of streptomycin. The cultures weremaintained at 37° C. in a humidified atmosphere containing 5% CO₂. Thecells were maintained as “monolayer culture” by serial sub-culturing.

Example 36 Methods for Cytotoxic Activities Against MCF7 and PC3: SRBCytotoxicity Assay

Cytotoxicity was determined using SRB method as previously described bySkehan et al. [Skehan P.; Storeng R.; Scudiero D.; Monks A.; McMahon J.;al., V. D. e., New colorimetric cytotoxicity assay for anticancer drugscreening. J Natl Cancer Inst 1990, 82, 1107-12, incorporated herein byreference in its entirety]. Exponentially growing cells were collectedusing 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000cells/well in RPMI-1640 supplemented medium. After 24 h, cells wereincubated for 72 h with various concentrations of the tested compounds.Following 72 h treatment, the cells would be fixed with 10%trichloroacetic acid for 1 h at 4° C. Wells were stained for 10 min atroom temperature with 0.4% SRB dissolved in 1% acetic acid. The plateswere air dried for 24 h and the dye was solubilized with Tris-HCl for 5min on a shaker at 1600 rpm. The optical density (OD) of each well wasmeasured spectrophotometrically at 564 nm with an ELISA microplatereader (ChroMate-4300, FL, USA). The IC₅₀ values were calculatedaccording to the equation for Boltzman sigmoidal concentration-responsecurve using the nonlinear regression fitting models (Graph Pad, PrismVersion 5).

Example 37 Methods for Experiments on HL-60 Leukemia Cell Lines: CellCulture and Reagents

HL-60 were purchased from CLS Cell Line Service GmbH (Eppelheim,Germany) and cultured in Roswell Park Memorial Institute-1640 medium(RPMI-1640; Thermo Fisher Scientific, Inc; Waltham, Mass., USA)supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific)and ciprofloxacin (10 μg/mL; Cipla Limited; Mumbai, India), at 37° C.with 5% CO₂ in a humidified incubator.

Example 38 Methods for Experiments on HL-60 Leukemia Cell Lines: CellViability Assay

The CellTiter®-Blue Cell Viability assay was acquired from PromegaCorporation (Madison, Wis., USA). Cell viability assay was performed asfollows. The cells (10⁴/well) were incubated with a concentrationgradient of KIM-161C and KIM-241C ranging from 0.01 to 100 μM, in96-well plates for 48 h at 37° C. Subsequently, 20 μL CellTiter®-BlueCell Viability reagent was added to each well and incubated for anadditional 2 h for the development of fluorescence. The fluorescenceemission was measured at 590 nm using the SpectraMax® i3x Multi-Modemicroplate reader (Molecular Devices, LLC; San Jose, Calif., USA.) andplotted against drug concentrations to determine the mean inhibitoryconcentration of KIM-161C and KIM-241C producing 50% decrease in cellviability (IC50).

Example 39 Methods for Experiments on HL-60 Leukemia Cell Lines: CellCycle Analysis

The cells (3.5×10⁵) were incubated with two different concentrations ofKIM-161C at 37° C. for 48 h. Subsequently, the cells were collected andwashed twice with ice-cold PBS (1×). The washed cells were fixed on icefor 20 min using a fixation buffer containing paraformaldehyde. Hoechst33342 (10 μg/mL; Thermo Fisher Scientific, Inc.) was used for staining.The cells were then incubated in the dark for 30 min on ice. A minimumtotal of 20,000 events were acquired using a BD FACSAria III flowcytometer. Flowlogic version 7.2.1 software (Inivai Technologies,Victoria, Australia) was used to obtain the percentages of cells in theG1, S, and G2/M phases in the singlet-gated population.

Example 40 Methods for Experiments on HL-60 Leukemia Cell Lines:Apoptosis Detection by Caspase 3 Activity

The caspase family of cysteine proteases are important regulators in theapoptosis. Caspase 3 belongs to the effector (caspase 3, 6 and 7) classof proteases and it is a key protease that is activated during earlystages of apoptosis. Like other proteases, caspase 3 is present in thecell as inactive zymogens that can be activated through proteolyticprocessing at conserved aspartic residue. The activated caspase 3 is amarker for cell apoptosis that proteolytically cleaves and activatesother caspases and intracellular targets [Patel, T.; Gores, G. J.;Kaufmann, S. H., The role of proteases during apoptosis. FASEB journal:official publication of the Federation of American Societies forExperimental Biology 1996, 10 (5), 587-97].

FITC conjugated active caspase-3 antibody (BD biosciences, USA) was usedto detect the active form of caspase 3 in the cells undergoingapoptosis. Briefly, cells were plated in a 6-well culture plate at adensity of 0.5×10⁶ cells and harvested after 24 hours of incubation withthe inhibitor treatment. The collected cells were washed twice in cold1×PBS and then re-suspended in a 0.5 mL BD Cytofix/Cytoperm solutionfollowed by 20 min incubation on ice. After incubation, cells werewashed twice in a BD Perm/Wash buffer (1×) and then labelled with 5 μLof FITC rabbit anti-caspase 3 antibodies. The labelled cells were washedagain with wash buffer and re-suspended in a 0.5 mL buffer and analyzedby acquiring a minimum of 5000 events on the FACS Aria III cell analyzerand sorter.

Example 41 Cytotoxic Assays Against MCF7 (Breast) and PC3 (Prostate)Cancer Cell Lines: Results

As discussed above, the compounds were screened for their anticanceractivities against variety of cell lines. Results are illustrated inTable 1.

TABLE 1 IC₅₀ values against MCF7 and PC3 MCF7 PC3 Code MWt IC₅₀ (μM)IC₅₀ (μM) KIM-111C 409.48 11.58 1.667 KIM-121C 443.93 20.82 6.86KIM-131C 455.5 9.48 5.83 KIM-161C 439.5 3.5 2.13 KIM-211C 471.54 34.8541.68 KIM-221C 505.99 44.25 74.16 KIM-231C 517.57 35.1 37.51 KIM-241C461.51 0.145 0.243 KIM-261C 501.57 32.07 38.68 KIM-111V 427.49 >100 >100KIM-131V 473.52 53.54 61.27 KIM-121V 443.92 >100 57.3 KIM-161V 457.5244.28 41.1 KIM-211V 489.56 >100 >100 KIM-221V 524 3.12 4.079 KIM-231V535.58 >100 >100 KIM-261V 519.59 >100 >100 KIM-2101V 521.58 >100 >100KIM-22101V 509.98 >100 >100 KIM-2216V 554.04 >100 >100 KIM-2230V513.97 >100 97.77 KIM-2245V 538.04 >100 >100 KIM-2246V 538.04 >100 >100KIM-2247V 538.04 >100 >100 KIM-2248V 542.00 89.63 85.11 KIM-2249V 600.1187.52 92.15 KIM-2250V 542.00 >100 >100 KIM-112V 294.35 >100 >100

Example 42 Cytotoxic Activity of KIM-161C and KIM-241C Against LeukemiaH60 Cell Lines and their Mechanism of Action

In order to confirm the activity of KIM-161C and KIM-241C, cellviability experiments were performed on HL-60 cells in the absence orpresence of a range of drug concentration gradients. KIM-161Cdemonstrated an IC₅₀ value of 262.5 nM, whereas the IC₅₀ value ofKIM-241C could not be obtained even at a concentration of 20 μM drug(FIG. 8). Therefore, it was decided that further investigations onmechanisms of cellular activities were conducted for KIM-161C only.

In order to understand the mechanism of action of KIM-161C, cell cycleanalysis of Hoechst 33342 stained HL-60 cells was performed using flowcytometry. HL-60 cells were first incubated with the IC₅₀ dose and the2×IC₅₀ doses of KIM-161C for 48 h. At the lower dose, i.e., 362.5 nM,KIM-161C led to an increase in cells in the G1 phase. KIM-161C at 725 nMled to a 3-fold increase in the cells in G2/M stage as compared to thecontrol. The G2/M arrest appeared to be mediated by anti-tubulinpolymerization properties of KIM-161C (FIGS. 9A-D).

HL-60 cells are known to be dependent on Src kinases fordifferentiation. Inhibition of Src kinase mediated activation of STAT3leads to apoptosis in HL-60 cells [Zhao, W. et al., orafenib inducesapoptosis in HL60 cells by inhibiting Src kinase-mediated STAT3phosphorylation, Anti-Cancer Drugs 2011, 22, 1, p 79-88, incorporatedherein by reference in its entirety]. We therefore performed apoptosisassay to detect caspase-3 activity (FIGS. 10A-G). Treatment of HL60cells with 362.5 nM of KIM-161C demonstrate more than 2-fold increase inthe cells undergoing apoptosis as compared to the basal apoptosis inHL-60 cells at both 24 and 48 h of treatment. Treatment of HL60 cellswith 725 nM of KIM-161C demonstrated more than 9.5-fold and 8.45-foldincrease in the cells undergoing apoptosis as compared to the basalapoptosis in HL-60 cells at both 24 and 48 h of treatment, respectively.

Example 43

In summary, two sets of compounds, 5-oxo-4,5-dihydro-1H-imidazol-1-yl(KIM-C series) and 3-aryl-2-acylaminopropenamido (KIM-V series)individually attached to N-benzylphenylacetamide pharmacophore, areprovided herein. Both sets of compounds were synthesized using similarsynthetic schemes that differ in the final step. Synthesized compoundswere confirmed by spectral analyses, and tested for their anticanceractivities against breast and prostate cell lines. The compoundseffectively inhibit cancer cell growth.

Mechanistic studies showed that these compounds affect cell division atthe mitosis stage and also promote apoptotic cell death. Therefore, itis likely that the disclosed compounds act via dual inhibition of Srckinase and tubulin. These compounds can be further developed forclinical applications in treating several types of solid and liquidtumors.

The invention claimed is:
 1. A compound of formula (I),

a salt thereof, a solvate thereof, a tautomer thereof, and astereoisomer thereof; wherein: R₁ is independently selected from thegroup consisting of an optionally substituted alkyl, an optionallysubstituted cycloalkyl, an optionally substituted arylalkyl, and anoptionally substituted aryl; R₂ is an optionally substituted aryl, or anoptionally substituted heteroaryl; R₃ is an optionally substituted aryl,or an optionally substituted heteroaryl; R₄ is selected from the groupconsisting of a hydrogen, an optionally substituted alkyl, an optionallysubstituted cycloalkyl, an optionally substituted arylalkyl, and anoptionally substituted aryl; R₅, and R₆ are independently selected fromthe group consisting of a hydrogen, an optionally substituted alkyl, anoptionally substituted cycloalkyl, and an optionally substitutedarylalkyl; R₇, R₈, R₉, and R₁₀ are independently selected from the groupconsisting of a hydrogen, an optionally substituted alkyl, an optionallysubstituted cycloalkyl, an optionally substituted arylalkyl, a hydroxy,and a halogen; and m is an integer in a range of 1-3.
 2. The compound ofclaim 1, wherein R₁ is methyl, or phenyl.
 3. The compound of claim 1,wherein R₂ is selected from the group consisting of phenyl,p-chlorophenyl, p-hydroxy-m-methoxyphenyl, p-methoxyphenyl, and2-furanyl.
 4. The compound of claim 1, wherein R₃ is phenyl.
 5. Thecompound of claim 1, wherein R₄ is hydrogen.
 6. The compound of claim 1,and wherein R₅ and R₆ are hydrogen.
 7. The compound of claim 1, whereinR₇, R₈, R₉ and R₁₀ are hydrogen.
 8. The compound of claim 1, which isselected from the group consisting of


9. A pharmaceutical composition, comprising: the compound of claim 1;and a pharmaceutically acceptable carrier and/or excipient.
 10. Thepharmaceutical composition of claim 9, wherein the pharmaceuticallyacceptable carrier and/or excipient is at least one selected from thegroup consisting of a buffer, an inorganic salt, a fatty acid, avegetable oil, a synthetic fatty ester, a surfactant, and a polymer. 11.The pharmaceutical composition of claim 9, wherein the compound isselected from the group consisting of